JP2024544071A - Use of one or more biomarkers to determine traumatic brain injury (TBI) in subjects with negative head computed tomography scan results for TBI - Google Patents

Abstract

Disclosed herein is a method for assisting in determining whether a subject has traumatic brain injury (TBI) by detecting the level of at least one biomarker, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in a sample taken from a subject, such as a human subject, where the subject has undergone a head CT scan with a negative result for TBI.

Description

This application claims priority to U.S. Patent Application No. 17/538,572, filed November 30, 2021, International Application No. PCT/US2021/061215, filed November 30, 2021, U.S. Provisional Application No. 63/284,421, filed November 30, 2021, and U.S. Provisional Application No. 63/294,271, filed December 28, 2021, the contents of each of which are incorporated herein by reference.

INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIAL The computer readable nucleotide/amino acid sequence listing, identified as follows, submitted contemporaneously herewith, is hereby incorporated by reference in its entirety: creation date November 29, 2022, file name "40136-601-SQL-ST26.xml", one 7.737 byte XML file.

The present disclosure relates to a method for aiding in the diagnosis and evaluation of a subject who has suffered, may have suffered, or is suspected of having suffered a head injury, such as a traumatic brain injury (TBI), comprising detecting a level of at least one biomarker, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in one or more samples taken from the subject at one or more time points within 24 hours of the time the subject suffered, may have suffered, or is suspected of having a head injury, the subject also undergoing a concurrent, prior, or subsequent (within a clinically relevant time frame) head computed tomography (CT) scan that is negative for TBI, and further comprising diagnosing the subject as likely to have TBI if the level of the one or more biomarkers in the sample is higher than a reference value.

Over 5 million mild traumatic brain injuries (TBIs) occur annually in the United States alone. Currently, there are no simple, objective, and accurate measurements available to aid in patient assessment. Indeed, much of TBI assessment and diagnosis is based on subjective data. Unfortunately, objective measurements such as head CT scans and the Glascow Coma Score (GCS) are not comprehensive or sensitive enough to assess mild TBI. Furthermore, head CT scans currently do not detect mild TBI in the majority of cases, are expensive, and expose patients to unnecessary radiation. Also, a negative head CT does not mean that the patient is no longer suspected of having a concussion, only that a given intervention, such as surgery, is not essential. Clinicians and patients need objective, reliable information to accurately assess this condition to facilitate appropriate triage and recovery. To date, there is limited data available to use UCH-L1 and GFAP in acute care settings to aid in patient assessment and management.

Mild TBI or concussion is very difficult to detect objectively and is a daily challenge in emergency rooms around the world. Concussion does not usually produce macroscopic findings such as hemorrhage or abnormalities on conventional computed tomography scans of the brain, but produces rapid neuronal dysfunction that resolves spontaneously over days to weeks. Approximately 15% of mild TBI patients suffer from persistent cognitive impairment. There is an unmet need for mild TBI victims in the field, emergency rooms and clinics, sports arenas, and military operations (e.g., combat).

Current algorithms for rating the severity of brain injury include the Glasgow Coma Scale score and other scales. Although these scales may be adequate for acute severity, they are insufficiently sensitive to subtle pathology, which may lead to persistent disability. The GCS and other scales also do not appear to be adequate because they cannot distinguish between types of injury. For example, patients enrolled in clinical trials and grouped into the same GCS level may have significant heterogeneity in injury severity and type. Inappropriate classification compromises the integrity of clinical trials, with corresponding variability in outcomes. Improved classification of injury would better distinguish disease severity and type in TBI patients in clinical trials.

In addition, current brain injury trials rely on outcome measures such as the Glasgow Outcome Scale Extended, which capture global phenomena but cannot assess subtle differences in outcomes. This has led to 30 consecutive failed trials of brain injury treatments.

Treatment and prevention trials require sensitive outcome measures to determine how well patients recover from brain injury.

In one aspect, the disclosure relates to an improved method for aiding in the diagnosis and evaluation of a subject, such as a human subject, who may have, or is suspected of having, a head injury. Such an improved method includes the steps of (1) performing, simultaneously or sequentially, an assay for measuring or detecting a level of a biomarker in a sample obtained from the subject within about 24 hours after an actual or suspected head injury, the biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, and (2) performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame, the improvement including diagnosing the subject as likely to have a traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and the head CT scan is negative for TBI.

In yet another aspect, the reference value is correlated with a cutoff value associated with: (a) a value in a subject suffering from a head injury; (b) occurrence of TBI in a subject; (c) stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) MRI positive rather than negative for TBI; (f) occurrence of amnesia in a subject (i.e., the presence or absence of amnesia); or (g) severity of TBI in a subject.

In another embodiment, the method further comprises the step of following up the subject for TBI if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI. In another embodiment, the method further comprises the step of administering treatment for TBI to the subject if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI. In yet another embodiment, the method further comprises the step of administering treatment for TBI to the subject and then following up the subject if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI.

In yet another aspect of the improved method, the sample can be collected within about 0 to about 12 hours after the actual or suspected head injury. For example, the sample can be collected within about 5 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 10 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 12 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 15 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 20 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 30 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 60 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 1.5 hours after the actual or suspected head injury. Alternatively, the sample may be collected within 2 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 3 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 4 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 5 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 6 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 7 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 8 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 9 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 10 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 11 hours of the actual or suspected head injury. Alternatively, the sample can be collected within 12 hours of the actual or suspected head injury.

In one embodiment, the improvement method is used to assess or evaluate the subject as having TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having mild TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having moderate TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having severe TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having moderate to severe TBI. In yet another embodiment, the improvement method is used to assess or evaluate the subject as not having TBI.

The improvement method can further include administering a TBI treatment (e.g., a surgical procedure, a therapeutic procedure, or a combination thereof) to a subject, such as a human subject, assessed or evaluated as having mild, moderate, severe, or moderate-severe TBI. Any such treatment known in the art and further described herein can be used. Additionally, in another embodiment, any subject being treated for TBI can optionally be followed during or after a course of treatment. Alternatively, the method can further include following up a subject (e.g., a subject that may not yet be receiving treatment) assessed as having mild, moderate, severe, or moderate-severe TBI.

In the above-mentioned improvement method, the sample can be selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. The blood sample can include a whole blood sample, a serum sample, or a plasma sample. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is a plasma sample. In still other embodiments, the sample is a serum sample. In still other embodiments, the sample is a saliva sample. In still other embodiments, the sample is an oropharyngeal sample. In still other embodiments, the sample is a nasopharyngeal sample. Such samples can be obtained in a variety of ways. For example, the sample can be obtained after the subject has experienced a head injury due to a body shake, a blunt impact from an external mechanical or other force resulting in a closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma. Alternatively, the sample can be obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, drugs of abuse, or one or more combinations thereof. Additionally, the sample can be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

The above-mentioned improvement method can be performed on any subject, such as a human subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory test values, the classification of the subject as having mild, moderate, severe, or moderate-severe TBI, whether the subject's UCH-L1, GFAP, and/or UCH-L1 and GFAP values are low, medium, or high, and the timing of events that caused or may have caused the subject to suffer a head injury.

In the above improved method, the assay is an immunoassay. In certain embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry test. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry test, and the sample is whole blood. In still other embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry test, and the sample is serum. In still other embodiments, the assay is a single molecule detection assay, and the sample is serum. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is plasma. In yet another embodiment, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In yet another embodiment, the assay is a clinical chemistry test, and the sample is plasma. In yet another embodiment, the assay is a single molecule detection assay, and the sample is plasma. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is saliva. In yet another embodiment, the assay is a point-of-care assay, the subject is a human, and the sample is saliva. In yet another embodiment, the assay is a clinical chemistry test, and the sample is saliva. In yet another embodiment, the assay is a single molecule detection assay, and the sample is saliva. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a clinical chemistry test, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal or nasopharyngeal sample.

In another aspect, the disclosure relates to a method for aiding in the diagnosis and evaluation of a subject, such as a human subject, having suffered, or potentially having suffered, or suspected of having suffered a head injury, the method comprising:
(1) an assay to measure or detect the level of a biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in a sample obtained from the subject within about 24 hours after actual or suspected head injury, and (2) a concurrent or sequential head computed tomography (CT) scan performed on the subject within a clinically relevant time frame;
b. if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI, diagnosing the subject as likely to have a traumatic brain injury (TBI).

In yet another aspect, the reference value is correlated with a cutoff value associated with: (a) a value in a subject suffering from a head injury; (b) occurrence of TBI in a subject; (c) stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) MRI positive rather than negative for TBI; (f) occurrence of amnesia in a subject (i.e., the presence or absence of amnesia); or (g) severity of TBI in a subject.

In another aspect, the method further comprises following up the subject for TBI if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI. In another aspect, the method further comprises administering treatment to the subject for TBI if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI. In yet another aspect, the method further comprises administering treatment to the subject for TBI and then following up the subject if the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI.

In yet another aspect of the method, the sample can be collected within about 0 to about 12 hours after the actual or suspected head injury. For example, the sample can be collected within about 5 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 10 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 12 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 15 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 20 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 30 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 60 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 1.5 hours after the actual or suspected head injury. Alternatively, the sample may be collected within 2 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 3 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 4 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 5 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 6 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 7 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 8 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 9 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 10 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 11 hours of the actual or suspected head injury. Alternatively, the sample can be collected within 12 hours of the actual or suspected head injury.

In one embodiment, the method is used to assess or evaluate the subject as having TBI. In another embodiment, the method is used to assess or evaluate the subject as having mild TBI. In another embodiment, the method is used to assess or evaluate the subject as having moderate TBI. In another embodiment, the method is used to assess or evaluate the subject as having severe TBI. In another embodiment, the method is used to assess or evaluate the subject as having moderate to severe TBI. In yet another embodiment, the method is used to assess or evaluate the subject as not having TBI.

The method can further include administering a TBI treatment (e.g., a surgical procedure, a therapeutic procedure, or a combination thereof) to a subject, such as a human subject, assessed or evaluated as having mild, moderate, severe, or moderate-severe TBI. Any such treatment known in the art and further described herein can be used. Additionally, in another embodiment, any subject being treated for TBI can optionally be followed during or after a course of treatment. Alternatively, the method can further include following up a subject (e.g., a subject that may not yet be receiving treatment) assessed as having mild, moderate, severe, or moderate-severe TBI.

In the above method, the sample can be selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a bodily fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. In certain embodiments, the sample is a whole blood sample. The blood sample can be a whole blood sample, a serum sample, or a plasma sample. In certain embodiments, the sample is a plasma sample. In still other embodiments, the sample is a serum sample. In still other embodiments, the sample is a saliva sample. In still other embodiments, the sample is an oropharyngeal sample. In still other embodiments, the sample is a nasopharyngeal sample. Such samples can be obtained in a variety of ways. For example, the sample can be obtained after the subject has experienced a head injury resulting from a body shake, a blunt impact from an external mechanical or other force resulting in a closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma. Alternatively, the sample can be obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, drugs of abuse, or one or more combinations thereof. Additionally, the sample can be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

The above method can be performed on any subject, such as a human subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory values, the classification of the subject as having suffered mild, moderate, severe, or moderate-severe TBI, whether the subject has low, moderate, or high UCH-L1, GFAP, and/or UCH-L1 and GFAP values, and the timing of events that caused or may have caused the subject to suffer a head injury.

In the above method, the assay is an immunoassay. In certain embodiments, the assay is a point-of-care assay. In still other embodiments, the assay is a clinical chemistry test. In still other embodiments, the assay is a single molecule detection assay. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In still other embodiments, the assay is a clinical chemistry test, and the sample is whole blood. In still other embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In still other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In still other embodiments, the assay is a clinical chemistry test, and the sample is serum. In still other embodiments, the assay is a single molecule detection assay, and the sample is serum. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is plasma. In yet another embodiment, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In yet another embodiment, the assay is a clinical chemistry test, and the sample is plasma. In yet another embodiment, the assay is a single molecule detection assay, and the sample is plasma. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is saliva. In yet another embodiment, the assay is a point-of-care assay, the subject is a human, and the sample is saliva. In yet another embodiment, the assay is a clinical chemistry test, and the sample is saliva. In yet another embodiment, the assay is a single molecule detection assay, and the sample is saliva. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a clinical chemistry test, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal or nasopharyngeal sample.

In yet another aspect, the disclosure relates to an improved method for aiding in the diagnosis and evaluation of a subject, such as a human subject, who may have or may have suffered a head injury. The improvement includes performing an assay to measure or detect a level of a biomarker in a sample obtained from the subject within about 24 hours after an actual or suspected head injury, the biomarker level being ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample, the improvement further includes diagnosing the subject as likely to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and a head computed tomography (CT) scan performed on the subject within a clinically relevant time frame is negative for TBI or the subject has not had a head CT scan performed.

In yet another aspect, the reference value is correlated with a cutoff value associated with: (a) a value in a subject suffering from a head injury; (b) occurrence of TBI in a subject; (c) stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) MRI positive rather than negative for TBI; (f) occurrence of amnesia in a subject (i.e., the presence or absence of amnesia); or (g) severity of TBI in a subject.

In another embodiment, the method further comprises the step of following up the subject for TBI if the value of the biomarker is higher than the reference value and, optionally, performing a head CT scan, the results of which are negative for TBI. In another embodiment, the method further comprises the step of administering treatment for TBI to the subject if the value of the biomarker is higher than the reference value and, optionally, performing a head CT scan, the results of which are negative for TBI. In yet another embodiment, the method further comprises the step of administering treatment for TBI to the subject and then following up the subject if the value of the biomarker is higher than the reference value and, optionally, performing a head CT scan, the results of which are negative for TBI.

In yet another aspect of the improved method, the sample can be collected within about 0 to about 12 hours after the actual or suspected head injury. For example, the sample can be collected within about 5 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 10 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 12 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 15 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within about 20 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 30 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 60 minutes after the actual or suspected head injury. Alternatively, the sample can be collected within 1.5 hours after the actual or suspected head injury. Alternatively, the sample may be collected within 2 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 3 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 4 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 5 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 6 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 7 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 8 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 9 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 10 hours of the actual or suspected head injury. Alternatively, the sample may be collected within 11 hours of the actual or suspected head injury. Alternatively, the sample can be collected within 12 hours of the actual or suspected head injury.

In one embodiment, the improvement method is used to assess or evaluate the subject as having TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having mild TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having moderate TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having severe TBI. In another embodiment, the improvement method is used to assess or evaluate the subject as having moderate to severe TBI. In yet another embodiment, the improvement method is used to assess or evaluate the subject as not having TBI.

The improvement method can further include administering a TBI treatment (e.g., a surgical procedure, a therapeutic procedure, or a combination thereof) to a subject, such as a human subject, assessed or evaluated as having mild, moderate, severe, or moderate-severe TBI. Any such treatment known in the art and further described herein can be used. Additionally, in another embodiment, any subject being treated for TBI can optionally be followed during or after a course of treatment. Alternatively, the method can further include following up a subject (e.g., a subject that may not yet be receiving treatment) assessed as having mild, moderate, severe, or moderate-severe TBI.

In the above-mentioned improvement method, the sample can be selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. The blood sample can be a whole blood sample, a serum sample, or a plasma sample. In certain embodiments, the sample is a whole blood sample. In certain embodiments, the sample is a plasma sample. In still other embodiments, the sample is a serum sample. In still other embodiments, the sample is a saliva sample. In still other embodiments, the sample is an oropharyngeal sample. In still other embodiments, the sample is a nasopharyngeal sample. Such samples can be obtained in a variety of ways. For example, the sample can be obtained after the subject has experienced a head injury due to a body shake, a blunt impact from an external mechanical or other force resulting in a closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma. Alternatively, the sample can be obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a chemical and a toxin. Examples of chemicals or toxins are mold, asbestos, pesticides, insecticides, organic solvents, paints, adhesives, gases, organometallics, drugs of abuse, or one or more combinations thereof. Additionally, the sample can be obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

The above-mentioned improvement method can be performed on any subject, such as a human subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory test values, the classification of the subject as having mild, moderate, severe, or moderate-severe TBI, whether the subject's UCH-L1, GFAP, and/or UCH-L1 and GFAP values are low, medium, or high, and the timing of events that caused or may have caused the subject to suffer a head injury.

In the above improved method, the assay is an immunoassay. In certain embodiments, the assay is a point-of-care assay. In yet other embodiments, the assay is a clinical chemistry test. In yet other embodiments, the assay is a single molecule detection assay. In yet other embodiments, the assay is an immunoassay, the subject is a human, and the sample is whole blood. In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is whole blood. In yet other embodiments, the assay is a clinical chemistry test, and the sample is whole blood. In yet other embodiments, the assay is a single molecule detection assay, and the sample is whole blood. In yet other embodiments, the assay is an immunoassay, the subject is a human, and the sample is serum. In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is serum. In yet other embodiments, the assay is a clinical chemistry test, and the sample is serum. In yet other embodiments, the assay is a single molecule detection assay, and the sample is serum. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is plasma. In yet another embodiment, the assay is a point-of-care assay, the subject is a human, and the sample is plasma. In yet another embodiment, the assay is a clinical chemistry test, and the sample is plasma. In yet another embodiment, the assay is a single molecule detection assay, and the sample is plasma. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is saliva. In yet another embodiment, the assay is a point-of-care assay, the subject is a human, and the sample is saliva. In yet another embodiment, the assay is a clinical chemistry test, and the sample is saliva. In yet another embodiment, the assay is a single molecule detection assay, and the sample is saliva. In yet another embodiment, the assay is an immunoassay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a point-of-care assay, the subject is a human, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a clinical chemistry test, and the sample is an oropharyngeal or nasopharyngeal sample. In yet other embodiments, the assay is a single molecule detection assay, and the sample is an oropharyngeal or nasopharyngeal sample.

In the above-mentioned improvement method, the reference value for GFAP is about 30 pg/mL to about 1700 pg/mL, and the reference value for UCH-L1 is about 150 pg/mL to about 700 pg/mL. In certain embodiments, the reference value for GFAP is about 90 pg/mL to about 1680 pg/mL, and the reference value for UCH-L1 is about 220 pg/mL to about 670 pg/mL. In other embodiments, the reference value for GFAP is about 110 pg/mL to about 950 pg/mL, and the reference value for UCH-L1 is about 160 pg/mL to about 320 pg/mL.

1A-D show ROC analyses of UCH-L1 or GFAP values correlated with Glasgow Coma Score (GCS) severity (i.e., GCS mild vs. moderate/severe) in subjects with negative CT scans for TBI. Samples were assessed within 12 hours of injury (FIGS. 1A, 1C) or within 24.1 hours of injury (FIGS. 1B, 1D). GFAP values are shown in FIGS. 1A and 1B. UCH-L1 values are shown in FIGS. 1C and 1D. 1A-D show ROC analyses of UCH-L1 or GFAP values correlated with Glasgow Coma Score (GCS) severity (i.e., GCS mild vs. moderate/severe) in subjects with negative CT scans for TBI. Samples were assessed within 12 hours of injury (FIGS. 1A, 1C) or within 24.1 hours of injury (FIGS. 1B, 1D). GFAP values are shown in FIGS. 1A and 1B. UCH-L1 values are shown in FIGS. 1C and 1D. 1A-D show ROC analyses of UCH-L1 or GFAP values correlated with Glasgow Coma Score (GCS) severity (i.e., GCS mild vs. moderate/severe) in subjects with negative CT scans for TBI. Samples were assessed within 12 hours of injury (FIGS. 1A, 1C) or within 24.1 hours of injury (FIGS. 1B, 1D). GFAP values are shown in FIGS. 1A and 1B. UCH-L1 values are shown in FIGS. 1C and 1D. 1A-D show ROC analyses of UCH-L1 or GFAP values correlated with Glasgow Coma Score (GCS) severity (i.e., GCS mild vs. moderate/severe) in subjects with negative CT scans for TBI. Samples were assessed within 12 hours of injury (FIGS. 1A, 1C) or within 24.1 hours of injury (FIGS. 1B, 1D). GFAP values are shown in FIGS. 1A and 1B. UCH-L1 values are shown in FIGS. 1C and 1D. Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-injury loss of consciousness (i.e., presence or absence) in subjects with CT scans negative for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or 24.1 hours (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F. Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-injury loss of consciousness (i.e., presence or absence) in subjects with CT scans negative for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or 24.1 hours (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F. Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-injury loss of consciousness (i.e., presence or absence) in subjects with CT scans negative for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or 24.1 hours (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F. Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-injury loss of consciousness (i.e., presence or absence) in subjects with CT scans negative for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or 24.1 hours (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F. Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-injury loss of consciousness (i.e., presence or absence) in subjects with CT scans negative for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or 24.1 hours (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F. Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-injury loss of consciousness (i.e., presence or absence) in subjects with CT scans negative for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or 24.1 hours (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F. Figures 3A-F show ROC analyses of UCH-L1 or GFAP values correlated with MRI results (i.e., positive or negative) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or 24.1 hours (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Figures 3A-F show ROC analyses of UCH-L1 or GFAP values correlated with MRI results (i.e., positive or negative) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or 24.1 hours (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Figures 3A-F show ROC analyses of UCH-L1 or GFAP values correlated with MRI results (i.e., positive or negative) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or 24.1 hours (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Figures 3A-F show ROC analyses of UCH-L1 or GFAP values correlated with MRI results (i.e., positive or negative) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or 24.1 hours (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Figures 3A-F show ROC analyses of UCH-L1 or GFAP values correlated with MRI results (i.e., positive or negative) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or 24.1 hours (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Figures 3A-F show ROC analyses of UCH-L1 or GFAP values correlated with MRI results (i.e., positive or negative) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or 24.1 hours (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or 24.1 hours (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F. Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or 24.1 hours (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F. Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or 24.1 hours (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F. Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or 24.1 hours (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F. Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or 24.1 hours (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F. Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or 24.1 hours (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F.

DETAILED DESCRIPTION The present disclosure relates to improved methods of aiding in the diagnosis and evaluation of a human subject having, potentially having, or suspected of having a head injury, such as a traumatic brain injury (e.g., mild, moderate, severe, or moderate-severe TBI), using biomarkers such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, optionally when the subject has undergone at least one head computed tomography (CT) scan within a clinically relevant time frame, the results of which are negative for TBI.

The methods described herein include detecting one or more biomarker values in one or more samples taken from a subject, such as a human subject, within about 24 hours of an actual or suspected head injury. Optionally, if a head CT scan is performed, it can be performed simultaneously or sequentially in any order, and can be performed simultaneously with, prior to, or after detecting one or more biomarker values in one or more samples taken from the subject. If the subject optionally undergoes a head CT scan, the results of which are negative for TBI, detecting one or more biomarker values, such as UCH-L1, GFAP, or a combination thereof, at a higher level than a reference value can facilitate accurately assessing or diagnosing a subject, such as a human subject, as more likely to have TBI. In other words, in certain embodiments, the improved methods described herein can identify subjects who have suffered a TBI but may have been erroneously diagnosed as not having a TBI based solely on the results of one or more head CT scans.

The section headings used in this section, and throughout the disclosure of this application, are for organizational purposes only and are not intended to be limiting.

1. Definitions Unless otherwise defined herein, scientific and technical terms used herein shall have the meaning commonly understood by those of ordinary skill in the art. In case of conflict, this application, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned in this application are incorporated herein by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not limiting.

As used herein, the terms "comprise," "include," "have," "have," "can," "contain," and variations thereof refer to open-ended phrases, terms, or words that do not exclude other acts or structures. Unless otherwise clear from the context, the singular indefinite articles, "and," and "a," "an," and "an" include plural references. Whether or not expressly stated, the present disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or components set forth herein.

When numerical ranges are specified in this application, each number within the range is specifically contemplated to the same precision. For example, in the range from 6 to 9, the numbers 7 and 8 are specifically contemplated in addition to 6 and 9, and in the range from 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are specifically contemplated.

The term "affinity matured antibody" is used herein to refer to an antibody that contains one or more alterations in one or more CDRs, which results in an improved affinity of the antibody for a target antigen (i.e., _KD , _kd , or k _a ) compared to a parent antibody that does not have the alterations. A typical affinity matured antibody will have nanomolar or picomolar affinity for the target antigen. Various methods for producing affinity matured antibodies are known in the art, including screening combinatorial antibody libraries generated using biodisplay methods. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., J. Immunol. 1999, 11:111-112 (1996); Random mutagenesis of CDR and/or framework residues is described in U.S. Pat. No. 6,914,128 B1, as well as in U.S. Pat. No. 6,914,128 B1, which describes selective mutations at selective mutagenesis positions and contact positions with activity enhancing amino acid residues or hypermutation positions.

As used herein, "analog assay" refers to an assay that measures the presence and/or concentration of an analyte in a test sample by measuring the total signal (e.g., fluorescence, color, etc.) generated by the analyte in the entire reaction mixture (e.g., a single reaction vessel). In an analog assay, noise cannot be distinguished from signal. One example of an analog assay is an assay that measures the presence and/or concentration of an analyte by measuring the total signal generated by multiple beads or microparticles contained in a single reaction vessel.

As used herein, "antibody" and "antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized antibodies), animal antibodies, such as, but not limited to, birds (e.g., ducks or geese), sharks, whales, and mammals, including non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, single chain Fvs ("scFvs"), single chain antibodies, single domain antibodies, Fab fragments, F(ab') fragments, F(ab') By antibodies is meant immunoglobulin _molecules , immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site, such as immunoglobulin molecules, disulfide-linked Fvs ("sdFvs"), and anti-idiotypic ("anti-Id") antibodies, dual domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual variable domain immunoglobulins and methods for their production are described in Wu, C., et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT Publication No. WO 2001/058956, the disclosures of each of which are incorporated herein by reference), as well as functionally active, epitope-binding fragments of any of the above. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, antibodies to an analyte are often referred to herein as "anti-analyte antibodies" or simply "analyte antibodies" (e.g., anti-UCH-L1 antibodies or UCH-L1 antibodies).

As used herein, "antibody fragment" refers to a portion of an intact antibody that contains an antigen binding site or variable region, said portion not including the heavy chain constant domain of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody idiotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab') ₂ fragments, Fd fragments, Fv fragments, diabodies, single chain Fv (scFv) molecules, single chain polypeptides comprising only one light chain variable domain, single chain polypeptides comprising the three CDRs of a light chain variable domain, single chain polypeptides comprising only one heavy chain variable region, and single chain polypeptides comprising the three CDRs of a heavy chain variable region.

"Area Under the Curve" or "AUC" means the area under the ROC curve. The AUC under the ROC curve is a measure of accuracy. If the AUC is 1, the test is perfect, if the AUC is 0.5, the test is useless. Preferred AUCs include at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990, or at least about 0.995.

"Beads" and "particles" are used interchangeably herein and refer to substantially spherical solid support. An example of a bead or particle is a microparticle. The microparticles that can be used herein can be of any type known in the art. For example, the beads or particles can be magnetic beads or particles. The magnetic beads/particles can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or magnetic fluid. Typical ferromagnetic materials include Fe, Co, Ni, Gd, Dy, _CrO2 , MnAs, MnBi, EuO, and NiO/ _Fe . Examples of _{ferrimagnetic} materials include _NiFe2O4 , _CoFe2O4 , _Fe3O4 (or _{FeO.Fe2O3 )} . The beads can have a magnetic solid core portion around which one or _more non-magnetic layers are disposed. Alternatively, the magnetic portion can be a layer around the non-magnetic core. The microparticles can be of any size that functions in the methods described herein, for example, from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

The term "binding protein" is used herein to refer to a monomeric or multimeric protein that binds to a binding partner, such as a polypeptide, an antigen, a compound or other molecule, or a substrate of any kind, to form a complex. A binding protein specifically binds to a binding partner. Binding proteins include antibodies and antigen-binding fragments thereof, as well as various other forms and derivatives thereof known in the art and described below, and other molecules that contain one or more antigen-binding domains that bind to an antigen molecule or a specific site (epitope) on an antigen molecule. Binding proteins therefore include, but are not limited to, antibodies, tetrameric immunoglobulins, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity matured antibodies, and fragments of any such antibodies that maintain antigen-binding ability.

The term "bispecific antibody" is used herein to refer to a full-length antibody produced by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or by knob-into-hole or similar approaches to introduce mutations into the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in multiple different immunoglobulin species, only one of which is a functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) in one of its two binding arms (a first HC/LC pair) and to another antigen (or epitope) in its second arm (another HC/LC pair). By this definition, bispecific antibodies have two antigen-binding arms that are distinct (both in specificity and CDR sequences) and are monovalent for each antigen to which they bind.

"CDR" is used herein to denote the "complementarity determining region" in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions. For each variable region, these regions are referred to as "CDR1", "CDR2", and "CDR3" from the N-terminus of the heavy or light chain, respectively. As used herein, the term "CDR set" refers to a set of three CDRs present in a single variable region that binds to an antigen. Thus, an antigen-binding site can contain six CDRs, including a CDR set from each of the heavy and light chain variable regions. A polypeptide containing one CDR (e.g., CDR1, CDR2, or CDR3) may also be referred to as a "molecular recognition unit". Crystal structure analysis of an antigen-antibody complex has demonstrated that the amino acid residues of the CDRs form extensive antigen contacts with the bound antigen, with the extensivest antigen contacts being with the heavy chain CDR3. Thus, it appears that the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site. In general, the CDR residues are directly and most substantially involved in affecting antigen binding.

The exact boundaries of these CDRs have been defined in various ways according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system that is applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are sometimes referred to as "Kabat CDRs." Chothia et al. (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) have described the Kabat CDRs as follows: It has been discovered that certain subportions within the CDRs have nearly identical peptide backbone structures despite great diversity at the amino acid sequence level. These subportions are referred to as "L1", "L2", and "L3", or "H1", "H2", and "H3", where "L" and "H" represent the light and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs", and their boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described in Padlan, FASEB J., 9:133-139 (1995) and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). While one may not strictly follow one of the systems described herein, it is possible to use the Kabat CDRs as a basis for determining which CDRs overlap with the Kabat CDRs. There are still other CDR boundary definitions that overlap with the CDRs, and may be shortened or lengthened in light of expectations or experimental results that certain residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use Kabat or Chothia defined CDRs.

"Clinically relevant time frame" means the time frame (e.g., seconds, minutes, or hours) required for a prudent and prudent medical professional (e.g., physician, nurse, paramedic, etc.) to reasonably determine that the results of one or more biomarker tests can be correlated to an imaging procedure such as a head CT scan, or a time frame that complies with guidelines established by an oversight body (e.g., a standards-setting body such as the World Health Organization (WHO), a Medical Council, or a regulatory approval authority such as the FDA, EMEA, etc.).

"Component", "components", or "at least one component" generally refers to capture antibodies, detection antibodies or conjugates, calibrators, controls, sensitivity panels, containers, buffers, diluents, salts, enzymes, enzyme cofactors, detection reagents, pretreatment reagents/solutions, substrates (e.g., in solution), stop solutions, etc. that may be included in a kit for assaying a test sample, such as a patient urine, whole blood, serum or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or may be lyophilized for reconstitution at the time of use in the assay.

As used herein, "correlated to" means compared to.

As used herein, "CT scan" refers to a computed tomography (CT) scan. A CT scan assembles a series of x-ray images taken from various angles and uses computer processing to create cross-sectional images or slices of bones, blood vessels, and soft tissues within the body. CT scans can use x-ray CT, positron emission tomography (PET), single photon emission computed tomography (SPECT), computer axial tomography (CAT scan), or computer assisted tomography. CT scans can be conventional CT scans or spiral/helical CT scans. Conventional CT scans scan slice by slice, for example from the top of the abdomen toward the pelvis, stopping after each slice and moving to the next slice. Conventional CT scans require the patient to hold their breath to avoid motion artifacts. Spiral/helical CT scans are continuous scans taken in a spiral, which is a significantly faster method and the scan images are continuous.

A head CT scan is "negative" for TBI when no intracranial lesion is found in images taken of a subject who may or may not have a head injury. More specifically, a subject's head CT scan is "negative" for TBI when no lesion is detected or confirmed, but the subject may still suffer from symptoms (e.g., of TBI) even if the head CT is negative. Because not all injuries or lesions can be visualized by head CT, most subjects will have a negative head CT for TBI. Thus, the methods and assays described herein can be used to provide an assessment or determination of subjects who have a negative head CT but may have TBI.

"Measured by an assay" is used herein to refer to the measurement of a reference value by any suitable assay. The measurement of the reference value can, in certain embodiments, be performed by the same type of assay as the assay to be applied to the sample from the subject (e.g., by immunoassays, clinical chemistry tests, single molecule detection assays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectrometric methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS)). The measurement of the reference value can, in certain embodiments, be performed by the same type of assay as the assay to be applied to the sample from the subject under the same assay conditions. As mentioned herein, the present disclosure provides exemplary reference values (e.g., calculated by comparing reference values at various time points). Based on the description provided by the present disclosure, it is well within the ability of a person of ordinary skill in the art to adapt the present disclosure to other assays to obtain assay-specific reference values for other assays. For example, a set of training samples including samples obtained from human subjects known to have suffered a head injury (more specifically, samples obtained from human subjects known to have suffered (i) mild TBI, and/or (ii) moderate, severe, or moderate-severe TBI) and samples obtained from human subjects known not to have suffered a head injury can be used to derive assay-specific reference values. It will be appreciated that a reference value "measured by an assay" and having a specified level of "sensitivity" and/or "specificity" is used herein to refer to a reference value determined to provide a specified sensitivity and/or specificity when said reference value is employed in the method of the invention. For example, it is well within the ability of one of ordinary skill in the art to determine the sensitivity and specificity associated with a given reference value in the method of the invention by repeated statistical analysis of assay data using multiple different possible reference values.

In fact, when distinguishing between subjects with traumatic brain injury and those without traumatic brain injury, or between subjects with mild traumatic brain injury and those with moderate, severe, or moderate-severe traumatic brain injury, one skilled in the art would weigh the impact of increasing the cutoff value on sensitivity and specificity. A higher or lower cutoff value would have a clear and predictable impact on sensitivity and specificity, as well as other standard statistical measures. It is well known that increasing the cutoff value may improve specificity but decrease sensitivity (the proportion of subjects who test positive for the disease). On the other hand, decreasing the cutoff value would improve sensitivity but decrease specificity (the proportion of subjects who test negative for the disease). The corresponding results of detecting traumatic brain injury or judging mild versus moderate, severe, or moderate-severe traumatic brain injury would be easily predicted by one skilled in the art. In distinguishing whether a subject has traumatic brain injury or not, or whether a subject has mild versus moderate, severe, or moderate-severe traumatic brain injury, a higher cutoff value improves specificity by distinguishing true negatives (i.e., subjects with no traumatic brain injury, subjects with no mild traumatic brain injury, subjects with no moderate traumatic brain injury, subjects with no severe traumatic brain injury, or subjects with no moderate-severe traumatic brain injury) from subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. However, at the same time, increasing the cutoff value reduces sensitivity by decreasing the total number of subjects identified as positive and the number of true positives. Conversely, a lower cutoff value improves sensitivity by distinguishing true positives (i.e., subjects with traumatic brain injury, subjects with mild traumatic brain injury, subjects with moderate traumatic brain injury, subjects with severe traumatic brain injury, or subjects with moderate to severe traumatic brain injury) from subjects without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate to severe traumatic brain injury. At the same time, however, lowering the cutoff value decreases specificity by increasing the total number of subjects identified as positive and the number of false positives.

In general, a high sensitivity value will allow a skilled artisan to more easily rule out a disease or condition (e.g., traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury), and a high specificity value will allow a skilled artisan to more easily diagnose a disease or condition. Whether a skilled artisan wants to rule out or confirm a disease depends on the patient outcome for each type of error. Thus, without full disclosure of the basic information on how the values were selected, it is not possible to know or predict the exact balance used to derive the test cutoff value. The balance of sensitivity and specificity and other factors will vary on a case-by-case basis. For this reason, it may be preferable in some cases to provide alternative cutoff (e.g., reference) values from which a physician or medical professional can choose.

As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications in its amino acid sequence compared to the true or parent antibody and that exhibits an altered domain structure. The derivative may have the typical domain structure found in a natural antibody and an amino acid sequence that is capable of specifically binding to a target (antigen). Typical examples of antibody derivatives are antibodies bound to other polypeptides, rearranged antibody domains, or antibody fragments. The derivative may further comprise at least one other compound, e.g. a protein domain, which is linked by covalent or non-covalent bonds. The linkage may be based on gene fusion according to methods known in the art. The additional domain present in the fusion protein comprising the antibody is preferably linked by a flexible linker, advantageously a peptide linker, which comprises a plurality of peptide-linked hydrophilic amino acids of sufficient length to accommodate the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody, or between the C-terminus of the antibody and the N-terminus of the additional protein domain. Antibodies can be linked to effector molecules having biological activity or structures suitable for selective binding to, for example, solid supports, bioactive agents (e.g., cytokines or growth hormones), chemicals, peptides, proteins, or drugs.

As used herein, "digital assay" refers to an assay that captures an analyte and separates and analyzes molecules of the analyte (e.g., to detect the presence and/or concentration of an analyte in a sample). In a digital assay, noise is separated from signal. In a digital assay, results are assigned a numerical value of 1 or 0. Examples of digital assays include one or more of the following assays (which may overlap, but are not mutually exclusive): single molecule detection assays, nanowell assays, single molecule enzyme immunoassays, direct capture and counting assays, etc.

The term "drug of abuse" is used herein to refer to one or more additional substances (e.g., drugs) taken for non-medical reasons (e.g., recreational and/or psychotropic effects). Excessive indulgence, use, or dependence on such drugs of abuse is often referred to as "substance abuse." Examples of drugs of abuse include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (e.g., ketamine, mescaline (peyote), PCP, psilocybin, DMT, and/or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances with psychotropic properties, such as, for example, nitrites, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

The term "dual specificity antibody" is used herein to refer to a full-length antibody capable of binding two different antigens (or epitopes) in each of its two binding arms (a pair of HC/LC) (see PCT Publication WO 02/02773). Thus, a dual specificity binding protein has two identical antigen-binding arms with the same specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

"Dual variable domain" is used herein to refer to two or more antigen binding sites on a binding protein, which may be a bivalent (two antigen binding sites), tetravalent (four antigen binding sites), or multivalent binding protein. A DVD may be monospecific, i.e., capable of binding one antigen (or one epitope), or multispecific, i.e., capable of binding two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). A preferred DVD binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig". Such DVD-Ig binding proteins are thus tetrameric and similar to, but provide more antigen binding sites than, an IgG molecule. That is, each half of a tetrameric DVD-Ig molecule is similar to half of an IgG molecule and contains a heavy chain DVD polypeptide and a light chain DVD polypeptide, but whereas the pair of heavy and light chains of an IgG molecule provides a single antigen-binding domain, the pair of heavy and light chains of a DVD-Ig provides two or more antigen-binding sites.

Each antigen binding site of a DVD-Ig binding protein can be derived from a donor ("parent") monoclonal antibody and therefore comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), with a total of six CDRs per antigen binding site involved in antigen binding. Thus, a DVD-Ig binding protein that binds two different epitopes (i.e., two different epitopes on two different antigen molecules or two different epitopes on the same antigen molecule) comprises an antigen binding site derived from a first parent monoclonal antibody and an antigen binding site derived from a second parent monoclonal antibody.

The design, expression and characterization of DVD-Ig binding molecules are described in PCT Publication No. WO2007/024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25:1290-1297 (2007). One preferred example of such a DVD-binding protein comprises a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker other than CH1, X2 is an Fc region, and n is 0 or 1, with 1 being preferred; and a light chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker other than CH1, X2 does not comprise an Fc region, and n is 0 or 1, with 1 being preferred. Such a DVD-Ig can include two such heavy chains and two such light chains, each chain including a variable domain linked in tandem without a constant region between the variable domains, the heavy and light chains can associate to form a tandem functional antigen binding site, and one pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein having four functional antigen binding sites. In another example, a DVD-Ig molecule can include heavy and light chains each including three variable domains (VD1, VD2, VD3) linked in tandem without a constant region between the variable domains, the heavy and light chains can associate to form three antigen binding sites, and the heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein having six antigen binding sites.

In a preferred embodiment, a DVD-Ig binding protein not only binds to the same target molecule as the binding partner of its parent monoclonal antibody, but also has one or more desirable properties of one or more of its parent monoclonal antibodies. Such additional properties are preferably one or more antibody parameters of the parent monoclonal antibodies. Antibody parameters that can be imparted to a DVD-Ig binding protein derived from one or more of its parent monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and orthologous antigen binding.

DVD-Ig binding proteins bind to at least one epitope of UCH-L1. Non-limiting examples of DVD-Ig binding proteins include DVD-Ig binding proteins that bind to one or more epitopes of UCH-L1, DVD-Ig binding proteins that bind to an epitope of human UCH-L1 and an epitope of UCH-L1 of another species (e.g., mouse), and DVD-Ig binding proteins that bind to an epitope of human UCH-L1 and an epitope of another target molecule.

As used herein, "dynamic range" refers to the range over which an assay readout is proportional to the amount of target molecule or analyte in the sample being analyzed.

"Epitope" or "epitopes" or "epitope of interest" means a site on any molecule that can be recognized and bind to a complementary site on its specific binding partner. The molecule and the specific binding partner form a specific binding pair. For example, an epitope can be present on a polypeptide, a protein, a hapten, a carbohydrate antigen (such as, but not limited to, a glycolipid, glycoprotein, or lipopolysaccharide), or a polysaccharide. The specific binding partner can be, but is not limited to, an antibody.

As used herein, "fragment antigen-binding fragment" or "Fab fragment" refers to a fragment of an antibody that binds to an antigen and contains one antigen-binding site, one complete light chain, and a portion of one heavy chain. Fab is a monovalent fragment composed of the VL, VH, CL, and CH1 domains. Fab is composed of one constant domain and one variable domain from each of the heavy and light chains. The variable domain contains a paratope (antigen-binding site) that contains a pair of complementarity determining regions at the amino terminus of the monomer. Thus, each arm of the Y binds to an epitope on the antigen. Fab fragments can be produced as described in the art, for example, using the enzyme papain, which can be used to cleave an immunoglobulin monomer into two Fab fragments and one Fc fragment, or they can be produced by recombinant means.

As used herein, "F(ab') ₂ fragment" refers to an antibody generated by pepsin digestion of a full-length IgG antibody to remove most of the Fc region while leaving a portion of the hinge region intact. F(ab') ₂ fragments are bivalent and have a molecular weight of about 110 kDa since they have two antigen-binding F(ab) portions linked together by disulfide bonds. Bivalent antibody fragments (F(ab') ₂ fragments) are shorter than full-length IgG molecules and are more likely to penetrate tissues, facilitating better antigen recognition in immunohistochemistry. The use of F(ab') ₂ fragments also avoids non-specific binding to Fc receptors or protein A/G on live cells. F(ab') ₂ fragments can bind and penetrate antigens.

As used herein, "framework" (FR) or "framework sequence" may refer to the remaining sequence of a variable region excluding the CDRs. The exact definition of a CDR sequence can be determined by various systems (see, for example, above), and the meaning of framework sequence is interpreted accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) further divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, with CDR1 being located between FR1 and FR2, CDR2 being located between FR2 and FR3, and CDR3 being located between FR3 and FR4. Unless a particular subregion is designated as FR1, FR2, FR3, or FR4, the framework region otherwise referred to refers to the entire FR within the variable region of a single native immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up a framework region.

Human heavy and light chain FR sequences are known in the art that can be used as heavy and light chain "acceptor" framework sequences (or simply "acceptor" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from framework sequences listed in public databases such as V-base (hypertext transfer protocol: //vbase.mrc-cpe.cam.ac.uk/) or the International ImMunoGeneTics® (IMGT®) information system (hypertext transfer protocol: //imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/).

As used herein, a "functional antigen-binding site" can refer to a site on a binding protein (e.g., an antibody) that is capable of binding to a target antigen. The antigen-binding affinity of the antigen-binding site need not be as strong as that of the parent binding protein (e.g., the parent antibody from which the antigen-binding site is derived), but the ability to bind to the antigen should be measurable using any one of a variety of methods known to assess proteins (e.g., antibodies that bind to antigens). Furthermore, the antigen-binding affinity of antigen-binding sites of multivalent proteins (e.g., multivalent antibodies) need not be quantifiable identical, as used herein.

"GFAP" is used herein to refer to glial fibrillary acidic protein. GFAP is a protein that is encoded by the GFAP gene in humans and can be made (e.g., by recombinant means in other species).

"GFAP status" can mean the value or amount of GFAP at a point in time (such as by a single measurement of GFAP), the value or amount of GFAP associated with follow-up (such as by repeated testing performed on a subject to determine whether the amount of GFAP increases or decreases), the value or amount of GFAP associated with treatment of traumatic brain injury (whether primary and/or secondary brain injury), or a combination thereof.

As used herein, "Glasgow Coma Scale" or "GCS" refers to a 15-point scale (described, for example, in 1974 by Graham Teasdale and Bryan Jennett, Lancet 1974;2:81-4) that provides a practical method for assessing impaired level of consciousness in patients who have suffered brain injury. This test measures best motor response, verbal response, and eye opening response with the following scores: I. Best motor response (6 points - complies with 2 partial requests; 5 points - moves hand over clavicle to neck stimulation; 4 points - bends elbow suddenly but shows no noticeable abnormality; 3 points - bends elbow and shows clearly noticeable abnormality; 2 points - straightens elbow; 1 point - no arm/leg movement, no interfering factor; NT - paralysis or other limiting factor); II. Verbal response (score 5 - correct name, place, and date; score 4 - disoriented but able to converse; score 3 - audible single word; score 2 - moaning/growls only; score 1 - no audible response, no distractors; NT - distractors disrupting speech); and III. Eye opening (score 4 - eyes open before stimulation; score 3 - after speaking or yelling; score 2 - after fingertip stimulation; score 1 - no eye opening at all, no distractors; NT - eyes closed due to local distractors). The final score is determined by adding the scores of I + II + III. If the GCS score is 13-15, the subject is considered to have mild TBI. If the GCS score is 9-12, the subject is considered to have moderate TBI. If the GCS score is 8 or less, typically 3-8, the subject is considered to have severe TBI.

As used herein, "Glasgow Outcome Scale" refers to a global scale of functional outcome that grades a patient's status into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery. "Glasgow Outcome Scale Extended" or "GOSE" is used interchangeably herein and provides a more detailed breakdown of the eight categories by subdividing the severe disability, moderate disability, and good recovery categories into lower and higher categories as shown in Table 1.

The term "humanized antibody" is used herein to refer to an antibody that contains heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and/or VL sequences have been altered to be "human-like", i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody or variant, derivative, analog, or fragment thereof that immunospecifically binds to an antigen of interest and contains a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. The term "substantially" as used herein with respect to CDRs refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') ₂ , FabC, Fv), with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions being those of a human immunoglobulin consensus sequence. In one embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. In some embodiments, the humanized antibody comprises a light chain and at least the variable domain of a heavy chain. The antibody may further comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody comprises only a humanized light chain. In some embodiments, the humanized antibody comprises only a humanized heavy chain. In certain embodiments, the humanized antibody comprises only a humanized variable domain of the light chain and/or only a humanized heavy chain.

Humanized antibodies can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can contain sequences from more than one class or isotype, and specific constant domains can be selected to optimize desired effector functions using techniques well known in the art.

The framework regions and CDRs of a humanized antibody need not correspond strictly to the parental sequences, and may be mutated in the donor antibody CDR or consensus framework, for example by substitution, insertion and/or deletion of at least one amino acid residue, such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in preferred embodiments, such mutations will not be extensive. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues will correspond to residues in the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. The term "consensus immunoglobulin sequence" as used herein means a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of cognate immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" can include "consensus framework regions" and/or "consensus CDRs". In a family of immunoglobulins, each position in a consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.

As used herein with respect to two or more polypeptide or polynucleotide sequences, "match" or "identity" can mean that the sequences have a specified percentage of residues that are identical over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage sequence identity. If the two sequences are of different length, or are misaligned at one or more ends when aligned such that only one of the sequences is included in the specified region being compared, the residues of the other sequence are included in the denominator of the calculation, but not in the numerator.

"Head injury" or "head injury" are used interchangeably herein and refer to any trauma to the scalp, skull, or brain. Such injuries may be simply a small bump on the skull or may be severe brain injuries. Such injuries include primary brain injury and/or secondary brain injury. Primary brain injury occurs at the time of initial injury and is due to the displacement of the brain's physical structures. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event and results in shearing and compression of the surrounding brain tissue. Secondary brain injury occurs after the primary injury and may involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop gradually over a period of time (hours to days) after the primary brain injury. This includes a whole cascade of cellular, chemical, tissue, or vascular changes in the brain, leading to further destruction of brain tissue.

Head injuries can be closed or open (penetrating). Closed head injuries refer to trauma to the scalp, skull, or brain where there is no penetration of the skull by the impacting object. Open head injuries refer to trauma to the scalp, skull, or brain where there is no penetration of the skull by the impacting object. Head injuries can result from bodily shaking by a person, blunt impact from external mechanical or other forces resulting in closed or open head injuries (e.g., vehicular accidents from automobiles, airplanes, trains, etc., blows to the head from baseball bats or firearms), cerebrovascular accidents (e.g., strokes), one or more falls (e.g., during athletic or other activities), explosions or blasts (collectively referred to as "blast injuries"), and other types of blunt force trauma. Alternatively, head injuries can result from inhalation and/or exposure to fire, chemicals, toxins, or combinations of chemicals and toxins. Examples of such chemicals and/or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometallics (such as methylmercury, tetraethyl lead, and organotins), and/or one or more drugs of abuse. Alternatively, the head injury may occur as a result of the subject suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2), fungal infection, bacterial infection, meningitis, hydrocephalus, or any combination thereof. In some cases, it may be uncertain whether such an event or injury has occurred or occurred. For example, the patient or subject may not have a medical history, may not be able to speak, or may not be aware of what events the subject has been exposed to. In such situations, the subject is said to be "possibly suffering from a head injury" in this application. In certain embodiments of this application, closed head injury does not include and specifically excludes cerebrovascular accidents such as strokes.

As used herein, "intracranial lesion" means an area of damage inside the brain. An intracranial lesion can be an abnormality seen on a brain imaging test, such as a CT scan or magnetic resonance imaging (MRI). On a CT or MRI scan, a brain lesion can appear as a dark or light spot that differs from normal brain tissue.

As used herein, an "isolated polynucleotide" can refer to a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, depending on its origin, is not associated with all or a portion of a polynucleotide with which it is naturally associated, is operably linked to a polynucleotide with which it is not naturally associated, or does not exist in nature as part of a larger sequence.

As used herein, "label" and "detectable label" refer to a moiety that is attached to an antibody or analyte to render the reaction of the antibody with the analyte detectable, and an antibody or analyte so labeled is said to be "detectably labeled." A label can produce a signal that is detectable by visual or instrumental means. Various labels include signal generators such as chromogens, fluorescent compounds, chemiluminescent compounds, and radioactive compounds. Representative examples of labels include moieties that produce light (e.g., acridinium compounds) and moieties that produce fluorescence (e.g., fluorescein). Other labels are described herein. In this regard, the moieties themselves may not be detectable, but may become detectable upon reaction with yet another moiety. Use of the term "detectably labeled" is intended to encompass such labels.

Any suitable detectable label known in the art can be used. For example, detectable labels include radioactive labels (e.g., 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), enzyme labels (e.g., horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, etc.), chemiluminescent labels (e.g., acridinium esters, thioesters, or sulfonamides, luminol, isoluminol, phenanthridinium esters, etc.), fluorescent labels, and the like. The label may be a label such as a fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dot (e.g., zinc sulfide capped cadmium selenide), thermometric label, or immunopolymerase chain reaction label. Guidance on labels, labeling procedures and detection of labels can be found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed. , Springer Verlag, N.Y. (1997) and in Molecular Probes, Inc., 1999. See Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), a handbook and catalog published by IEEE, Inc., Eugene, Oregon. Fluorescent labels can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, the disclosures of which are incorporated herein in their entireties). In homogeneous chemiluminescent assays, acridinium compounds can be used as detectable labels (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4:2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett. 14:3917-3921 (2004); and Adamczyk et al., Org. Lett. 5:3779-3782 (2003)).

In one embodiment, the acridinium compound is acridinium-9-carboxamide. Methods for producing acridinium-9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin. 6:107-114 (1991); Adamczyk et al., J. Org. Chem. 63:5636-5639 (1998); Adamczyk et al., Tetrahedron 55:10899-10914 (1999); Adamczyk et al., Org. Lett. 1:779-781 (1999); Adamczyk et al. , Bioconjugate Chem. 11:714-724 (2000); Mattingly et al., In Luminescence Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al., Org. Lett. 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699 (each of which is incorporated herein by reference in its entirety for the teachings thereof).

Another example of an acridinium compound is acridinium-9-carboxylic acid aryl ester. One example of an acridinium-9-carboxylic acid aryl ester of formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylic acid aryl esters are described in McCapra et al., Photochem. Photobiol. 4:1111-21 (1965); Razavi et al., Luminescence 15:245-249 (2000); Razavi et al., Luminescence 15:245-249 (2000); , Luminescence 15:239-244 (2000), and U.S. Pat. No. 5,241,070, each of which is incorporated herein by reference in its entirety for the teachings of said methods. Such acridinium-9-carboxylic acid aryl esters are efficient chemiluminescent indicators, in terms of signal intensity and/or signal speed, of hydrogen peroxide generated upon oxidation of an analyte by at least one oxidase molecule. The chemiluminescent process of acridinium-9-carboxylic acid aryl esters is completed rapidly (i.e., in less than one second), whereas acridinium-9-carboxamide chemiluminescence takes more than two seconds. On the other hand, acridinium-9-carboxylic acid aryl esters lose their chemiluminescence in the presence of proteins. Thus, their use requires the absence of proteins during signal generation and detection. Methods for separating or removing proteins in a sample are well known to those of skill in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and/or digestion (see, e.g., Wells, High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies, Elsevier (2003)). The amount of protein removed or separated from a test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Additional details regarding acridinium-9-carboxylic acid aryl esters and their uses are described in U.S. patent application Ser. No. 11/697,835, filed Apr. 9, 2007. The acridinium-9-carboxylic acid aryl ester can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

"Linking sequence" or "linking peptide sequence" refers to a naturally occurring or artificial polypeptide sequence that is linked to one or more polypeptide sequences of interest (e.g., full length, fragments, etc.). The term "linked" refers to the association of the linking sequence with the polypeptide sequence of interest. Preferably, such polypeptide sequences are linked by one or more peptide bonds. Linking sequences can be from about 4 to about 50 amino acids in length. Linking sequences are preferably from about 6 to about 30 amino acids in length. Natural linking sequences can be modified by amino acid substitution, addition, or deletion to create artificial linking sequences. Linking sequences can be used in many applications, including use in recombinant Fabs. Exemplary linking sequences include, but are not limited to: (i) Histidine (His) tags, such as a 6xHis tag having an amino acid sequence of HHHHHH (SEQ ID NO: 3), are useful as linking sequences to facilitate isolation and purification of polypeptides and antibodies of interest. (ii) Enterokinase cleavage sites, like His tags, are used to isolate and purify proteins and antibodies of interest. Often, enterokinase cleavage sites are used in conjunction with His tags in isolating and purifying proteins and antibodies of interest. Various enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence DDDDK (SEQ ID NO: 4) and derivatives thereof, such as ADDDDK (SEQ ID NO: 5). (iii) Other sequences can also be used to link or join the light and/or heavy chain variable regions of the single chain variable region fragment. Examples of other linking sequences are described in Bird et al., Science 242:423-426 (1988); Huston et al., PNAS USA 85:5879-5883 (1988); and McCafferty et al. , Nature 348:552-554 (1990). The linking sequence can also be modified for additional functions, such as drug attachment or attachment to a solid support. In the context of the present disclosure, the monoclonal antibody can include a linking sequence, such as, for example, a His tag, an enterokinase cleavage site, or both.

"Magnetic Resonance Imaging" or "MRI" are used interchangeably herein and refer to a medical imaging technique used in radiology to produce images of living anatomical structures and physiological processes in both health and disease (e.g., interchangeably referred to herein as "MRI", "MRI procedure" or "MRI scan"). MRI is a medical imaging technique that measures the response of atomic nuclei in living tissue to high frequency radio waves when placed in a strong magnetic field, producing images of internal organs. MRI scanners are based on the science of nuclear magnetic resonance (NMR) and use strong magnetic fields, radio waves and magnetic field gradients to produce images inside the body.

As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible minor natural mutations. Monoclonal antibodies are highly specific for a single antigen. Moreover, in contrast to polyclonal antibody preparations, which generally contain different antibodies against different determinants (epitopes), each monoclonal antibody is specific for a single determinant on the antigen. Monoclonal antibodies in this application specifically include "chimeric" antibodies in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, and the remainder of the antibody chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies so long as they exhibit the desired biological activity.

The term "multivalent binding protein" is used herein to refer to a binding protein that contains two or more antigen binding sites (also referred to herein as "antigen binding domains"). Multivalent binding proteins are preferably artificially created to have three or more antigen binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein that can bind to two or more related or unrelated targets, including binding proteins that can bind to two or more different epitopes of the same target molecule.

"Negative predictive value" or "NPV" are used interchangeably herein and refer to the probability that a subject outcome will be negative given a subject test result that is negative.

As used herein, a "reference value" refers to an assay cutoff value used to assess diagnosis, prognosis, or therapeutic efficacy, and which is conventionally or herein associated with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement, etc.). As used herein, an "absolute amount" refers to the absolute value of the change or difference between at least two assay results taken or sampled at different time points, which, like a reference value, is a numerical value conventionally or herein associated with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement, etc.). As used herein, an "absolute value" refers to a real numerical value (e.g., the difference between two compared values (e.g., a value measured at a first time point and a value measured at a second time point)) regardless of its sign, i.e., positive or negative.

The present disclosure provides exemplary reference values and absolute amounts (e.g., calculated by comparing reference values at different time points). However, it is well known that the reference values and absolute amounts may vary depending on the type of immunoassay (e.g., the antibody used, the reaction conditions, the sample purity, etc.), and the assays can be compared and standardized. Furthermore, based on the description provided by the present disclosure, it is well within the ability of a person of ordinary skill in the art to adapt the present disclosure to other immunoassays to obtain immunoassay-specific reference values and absolute amounts for other immunoassays. Although the exact values of the reference values and absolute amounts may vary between assays, the findings described herein are generally applicable and can be extrapolated to other assays.

"Point-of-care device" means a device used to provide medical diagnostic testing at or near the point of care (i.e., outside a laboratory) at the site of patient care (e.g., a hospital, doctor's office, emergency or other medical care facility, the patient's home, a nursing home and/or a long-term care and/or hospice facility). Examples of point-of-care devices include products from Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity), Universal Biosensors (Rowville, Australia) (see US 2006/0134713), Axis-Shield PoC AS (Oslo, Norway) and Clinical Lab Products (Los Angeles, USA).

"Positive predictive value" or "PPV" are used interchangeably herein and refer to the probability that a subject outcome will be positive given a subject test result.

"Quality control reagents" for the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. A (e.g., one or more, such as multiple) "calibrator" or "standard" is typically used to generate a calibration (standard) curve for the interpolation of the concentration of an analyte, such as an antibody or analyte. Alternatively, a single calibrator near a reference or control value (e.g., a "low", "medium", or "high" value) can be used. Multiple calibrators (i.e., two or more calibrators or calibrators with varying amounts) can be used in combination to form a "sensitivity panel."

"Receiver Operating Characteristic" curve or "ROC" curve refers to a graphical plot showing the performance of a binary classifier system as its discrimination threshold varies. For example, an ROC curve can be a plot of true positive rate against false positive rate for various possible cutoff points of a diagnostic test. The curve is constructed by plotting the proportion of true positives among positives (TPR=true positive rate) against the proportion of false positives among negatives (FPR=false positive rate) at various threshold settings. TPR is also called sensitivity, and FPR is 1-specificity or true negative rate. ROC curves show a trade-off between sensitivity and specificity (increasing sensitivity is accompanied by decreasing specificity), with a test becoming more accurate as the curve approaches the left end of ROC space and then the top end, and a test becoming less accurate as the curve approaches the 45 degree diagonal of ROC space, with the slope of the tangent at the cutoff point representing the likelihood ratio (LR) of that value of the test, and the area under the curve being a measure of the accuracy of the test.

By "recombinant antibody" and "recombinant antibodies" is meant an antibody produced by one or more steps including recombinantly cloning a nucleic acid sequence encoding all or part of one or more monoclonal antibodies into a suitable expression vector and then expressing said antibodies in a suitable host cell. The term includes, but is not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized antibodies), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Ig(R), and other antibodies described in (i) herein (dual variable domain immunoglobulins and methods for their production are described in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)). As used herein, the term "bifunctional antibody" refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificity.

As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., a patient) refers to the evaluation of factors, including biomarkers, to predict the risk of occurrence of a future event, including disease onset or disease progression, so that more informed treatment decisions can be made regarding the subject.

The terms "sample," "test sample," "specimen," "sample from a subject," "biological sample," and "patient sample" are used interchangeably herein and may refer to whole blood (including, e.g., capillary blood, venous blood, dried blood spots, etc.), blood such as serum or plasma, tissue, saliva, urine, amniotic fluid, oropharyngeal specimen, nasopharyngeal specimen, lower respiratory tract specimen such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage fluid, cerebrospinal fluid, placental cells or tissue, vascular endothelial cells, leukocytes, or monocyte samples. The sample may be used directly as obtained from the patient or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc. to modify the characteristics of the sample in any manner described herein or otherwise known in the art. Additionally, the sample can be a nasopharyngeal or oropharyngeal sample obtained using one or more swabs, which are then stored in a sterile tube containing viral transport medium (VTM) or universal transport medium (UTM) after collection, or transferred to another testing medium.

A variety of cell types, tissues, or bodily fluids can be used to obtain samples. Such cell types, tissues, and bodily fluids include tissue sections such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, frozen sections taken for histological examination, blood (e.g., whole blood, dried blood spots, etc.), plasma, serum, saliva, red blood cells, platelets, interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues can also include lymphatic fluid, cerebrospinal fluid, or any bodily fluid taken by aspiration. Tissues or cell types can be provided by taking cell samples from humans and non-human animals, or can be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those with treatment or outcome history, can be used. Protein or nucleotide isolation and/or purification may not be required. In certain embodiments, the sample is a blood sample (e.g., a whole blood sample, a serum sample, or a plasma sample). In certain embodiments, the sample is a whole blood sample. In some embodiments, the sample is a capillary blood sample. In some embodiments, the sample is a dried blood spot. In some embodiments, the sample is a serum sample. In still other embodiments, the sample is a plasma sample. In some embodiments, the sample is an oropharyngeal specimen. In other embodiments, the sample is a nasopharyngeal specimen. In other embodiments, the sample is sputum. In other embodiments, the sample is an endotracheal aspirate. In still other embodiments, the sample is bronchoalveolar lavage fluid. In still other embodiments, the sample is a saliva sample.

As used herein, the "sensitivity" of an assay refers to the proportion of subjects that have a positive outcome and are correctly identified as positive (e.g., correctly identifying subjects that have the disease or condition targeted by the test). For example, this can include correctly identifying subjects with TBI from subjects without TBI, correctly identifying subjects with moderate, severe, or moderate-severe TBI from subjects with mild TBI, correctly identifying subjects with mild TBI from subjects with moderate, severe, or moderate-severe TBI, correctly identifying subjects with moderate, severe, or moderate-severe TBI from subjects without TBI, or correctly identifying subjects with mild TBI from subjects without TBI.

As used herein, the "specificity" of an assay refers to the proportion of subjects who have a negative outcome and are correctly identified as negative (e.g., correctly identifying subjects who do not have the disease or condition targeted by the test). For example, this can include correctly identifying subjects who do not have TBI from subjects who have TBI, correctly identifying subjects who do not have moderate, severe, or moderate-severe TBI from subjects who have mild TBI, correctly identifying subjects who do not have mild TBI from subjects who have moderate, severe, or moderate-severe TBI, etc.

"Calibration composition series" means a number of compositions containing known concentrations of UCH-L1, each of which differs from the other compositions in the series in the concentration of UCH-L1.

"Solid phase" or "solid support" are used interchangeably herein and refer to any material that can be used to bind and/or attract and immobilize (1) one or more capture agents or capture specific binding partners, or (2) one or more detection agents or detection specific binding partners. A solid phase can be selected for its inherent ability to attract and immobilize a capture agent. Alternatively, the solid phase can be coupled to a linking agent that has the ability to attract and immobilize (1) a capture agent or capture specific binding partner, or (2) a detection agent or detection specific binding partner. For example, the linking agent can include a charged substance that is oppositely charged relative to the capture agent (e.g., capture specific binding partner) or detection agent (e.g., detection specific binding partner) itself, or to a charged substance bound to (1) the capture agent or capture specific binding partner, or (2) the detection agent or detection specific binding partner. In general, the linking agent can be any binding partner that can be immobilized (bound) to a solid phase and, through a binding reaction, immobilize (preferably specific) (1) a capture agent or a specific binding partner for capture, or (2) a detection agent or a specific binding partner for detection. The linking agent allows the capture agent to be indirectly bound to the solid phase material before or during the performance of the assay. For example, the solid phase can be made of plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips, and other structures known to those of ordinary skill in the art.

As used herein, "specific binding" or "specifically binds" can refer to an interaction between an antibody, protein, or peptide and a second chemical species that is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species, e.g., an antibody recognizes and binds to a particular protein structure rather than proteins in general. If an antibody is specific for epitope "A", then the inclusion of a molecule containing epitope A (free, unlabeled A) in a reaction involving labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

A "specific binding partner" is a member of a specific binding pair. A specific binding pair comprises two distinct molecules that specifically bind to each other by chemical or physical means. Thus, in addition to the typical immunoassay antigen-antibody specific binding pair, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like. Additionally, specific binding pairs can include members that are analogs of the original specific binding member (e.g., analyte analogs). Immunoreactive specific binding members include antigens, antigen fragments, and antibodies, including monoclonal and polyclonal antibodies, whether isolated or recombinantly produced, as well as complexes and fragments thereof.

As used herein, "statistically significant" refers to the probability that a relationship between two or more variables is due to causes other than chance. Statistical hypothesis testing is used to determine whether the results of a data set are statistically significant. In statistical hypothesis testing, a result is statistically significant if the observed p-value of the test statistic is less than the significance level established for the test. The p-value is the probability of obtaining a result that is at least as deviant from the hypothesis as the observed result, assuming that the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon signed rank test, t-test, chi-square test, or Fisher's exact test. As used herein, "significant" refers to a change that has not been determined to be statistically significant (e.g., a statistical hypothesis test may not have been performed).

The terms "subject" and "patient" are used interchangeably herein and refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus and rhesus monkeys, chimpanzees, etc.), and humans). In certain embodiments, the subject may be human or non-human. In certain embodiments, the subject is human. The subject or patient may also be undergoing other types of treatment.

"Treat", "therapeutic" or "treatment" are used interchangeably herein and refer to reversing, alleviating or inhibiting the progression of the disease and/or injury to which such term applies, or one or more symptoms of such disease. Depending on the condition of the subject, the term may also refer to preventing the disease, including preventing the onset of the disease or preventing symptoms associated with the disease. Treatment may be performed acutely or chronically. The term may further refer to reducing the severity of the disease or symptoms associated with such disease prior to onset of the disease. Such prevention or reduction of the severity of a disease prior to onset refers to administering the pharmaceutical composition to a subject not suffering from the disease at the time of administration. "Prevent" may also refer to preventing the recurrence of the disease or one or more symptoms associated with such disease. "Treatment" and "therapeutic" refer to the act of treatment, and "treating" is as defined above.

"Traumatic brain injury" or "TBI" are used interchangeably herein and refer to a complex injury with a wide range of symptoms and disabilities. TBI, like other injuries, is most often an acute event. TBI can be classified as "mild," "moderate," or "severe." Causes of TBI are diverse and include, for example, shaking by a person, motor vehicle accidents, fire injuries, cerebrovascular accidents (e.g., strokes), falls, explosions or blasts, and other types of blunt force trauma. Other causes of TBI include inhalation and/or exposure to one or more fires, chemicals or toxins (e.g., mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (e.g., carbon monoxide, hydrogen sulfide, and cyanides), organometallics (e.g., methylmercury, tetraethyl lead, and organotins), one or more drugs of abuse, or combinations thereof. Alternatively, TBI may occur in subjects suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2, meningitis, etc.), fungal infection (e.g., meningitis), bacterial infection (e.g., meningitis), or any combination thereof. Age groups at highest risk for TBI are young adults and the elderly. In certain embodiments of the present application, traumatic brain injury or TBI does not include and specifically excludes cerebrovascular accidents, such as stroke.

As used herein, "mild TBI" refers to a head injury in which the subject may or may not lose consciousness. Loss of consciousness in a subject is generally brief, usually only for a few seconds or minutes. Mild TBI is also referred to as concussion, minor head trauma, minor TBI, minor brain injury, and minor head injury. While MRI and CT scans are often normal, individuals with mild TBI may have cognitive impairment such as headaches, impaired thinking, memory problems, mood swings, and irritability.

Mild TBI is the most common type of TBI and is often overlooked at the time of initial injury. Typically, subjects have a Glasgow Coma Scale score of 13-15 (e.g., 13-15 or 14-15). 15% of people with mild TBI have symptoms that last for more than 3 months. Common symptoms of mild TBI include fatigue, headache, visual disturbances, memory loss, attention/concentration problems, sleep problems, dizziness/balance problems, irritability-emotional disorders, depressed feelings, and seizures. Other symptoms associated with mild TBI include nausea, decreased sense of taste, sensitivity to light and sound, mood swings, feeling lost or confused, and/or slowed thinking.

As used herein, "moderate TBI" refers to brain injury resulting in loss of consciousness and/or confusion and disorientation for 1-24 hours and a subject's Glasgow Coma Scale score of 9-13 (e.g., 9-12 or 9-13). Individuals with moderate TBI may have abnormal brain imaging results. As used herein, "severe TBI" refers to brain injury resulting in loss of consciousness for more than 24 hours, memory loss after injury or penetrating cranial injury for more than 24 hours, and a subject's Glasgow Coma Scale score of 3-8. Impairments range from higher cognitive impairment to coma. Recovery may leave individuals with reduced arm or leg function, speech or language abnormalities, reduced thinking ability, or emotional impairment. Individuals with severe injury may remain unresponsive for extended periods of time. Many individuals with severe TBI often require long-term rehabilitation to maximize function and independence.

As used herein, "moderate-severe" TBI refers to a range of brain injury including changes from moderate to severe TBI over time, and thus encompassing (e.g., temporary) moderate TBI alone, severe TBI alone, and moderate-severe TBI combined. For example, in some clinical situations, a subject may initially be diagnosed with moderate TBI, but over time (minutes, hours, or days), progress to severe TBI (e.g., as in the setting of a cerebral hemorrhage). Alternatively, in some clinical situations, a subject may initially be diagnosed with severe TBI, but over time (minutes, hours, or days), transition to moderate TBI. Such subjects are examples of patients who can be classified as "moderate-severe." Common symptoms of moderate to severe TBI include problems with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing, and/or "executive functioning", inability to understand what others are saying (receptive aphasia), difficulty speaking and being understood by others (expressive aphasia), slurred speech, speaking very quickly or very slowly, difficulty reading, difficulty writing, difficulty interpreting touch, temperature, movement, limb position, and fine discrimination, integration or patterning of sensory impressions into psychologically meaningful data, complete or partial loss of vision, eye muscle weakness and double vision (diplopia), blurred vision, difficulty judging distances, These include involuntary eye movements (nystagmus), light intolerance (photophobia), hearing impairments such as reduced or lost hearing, tinnitus (ringing in the ears), increased sensitivity to sound, loss or diminished sense of smell (anosmia), loss or diminished sense of taste, convulsions associated with epilepsy which can be classified into several types and may involve disruptions in consciousness, sensory perception or movement, impaired bowel and bladder control, sleep disorders, decreased stamina, appetite changes, impaired thermoregulation, menstrual difficulties, addictive behaviors, impaired emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or cognitive impairment including denial/lack of awareness. Subjects with moderate to severe TBI can have a Glasgow Coma Scale score of 3-12 (including moderate TBI scores of 9-12 and severe TBI scores of 3-8).

"Ubiquitin carboxy-terminal hydrolase L1" or "UCH-L1" are used interchangeably herein and refer to the deubiquitinating enzyme encoded in humans by the UCH-L1 gene. UCH-L1, also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to yield ubiquitin monomers.

"UCH-L1 status" can mean the value or amount of UCH-L1 at a point in time (such as by a single measurement of UCH-L1), the value or amount of UCH-L1 associated with follow-up (such as by repeated testing performed on a subject to determine whether the amount of UCH-L1 increases or decreases), the value or amount of UCH-L1 associated with treatment of traumatic brain injury (whether primary and/or secondary brain injury), or a combination thereof.

"Variants" are used herein to refer to peptides or polypeptides that differ in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but that maintain at least one biological activity. Representative examples of "biological activity" include the ability to bind a specific antibody or promote an immune response. Variants are also used herein to refer to proteins having an amino acid sequence that is substantially identical to a reference protein having an amino acid sequence that maintains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with another amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are generally recognized in the art as involving minor changes. These minor changes can be identified, in one aspect, by considering the hydropathic index of the amino acid as understood in the art. See Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted while maintaining protein function. In one embodiment, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that maintain biological function. Considering the hydrophilicity of amino acids in relation to a peptide, the maximum local average hydrophilicity of the peptide can be calculated, although the maximum local average hydrophilicity is a useful tool that has been reported to correlate well with antigenicity and immunogenicity. See U.S. Pat. No. 4,554,101, which is incorporated herein by reference in its entirety. As understood in the art, substitutions of amino acids with similar hydrophilicity values result in peptides that maintain biological activity (e.g., immunogenicity). Substitutions can be made with amino acids whose hydrophilicity values are within ±2 of each other. Both the hydrophobic index and hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are believed to depend on the relative similarity of amino acids, particularly the side chains of those amino acids, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" can also be used to refer to an antigenically reactive fragment of an anti-UCH-L1 antibody that differs in amino acid sequence from the corresponding fragment of the anti-UCH-L1 antibody, but is still antigenically reactive and can compete with the corresponding fragment of the anti-UCH-L1 antibody for binding to UCH-L1. "Variant" can also be used to refer to a polypeptide or fragment thereof that has been differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, but that retains its antigenic reactivity.

"Vector" is used herein to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One example of a vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, and thus are replicated along with the host genome. Additionally, certain vectors can induce the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most widely used form of vector, "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses) that perform equivalent functions can also be used. In this regard, RNA vectors (including RNA viral vectors) are also applicable in the context of the present disclosure.

Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. For example, all nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are well known and widely used in the art. The meaning and scope of the terms should be clear, but in case of potential ambiguity, the definitions set forth in this application take precedence over any dictionary or extrinsic definitions. Furthermore, unless necessarily otherwise from the context, singular terms include the plural and plural terms include the singular.

2. Methods to Help Determine Traumatic Brain Injury (TBI) in Subjects with Negative Head Computed Tomography (CT) Scan Results The present disclosure relates to methods to help determine whether a subject is more likely to have suffered a traumatic brain injury (TBI), such as a human subject who has suffered, may have suffered, or is suspected of having suffered a head injury, when the subject has undergone one or more head CT scans, the results of which are negative for TBI. Specifically, such methods can include (a) (1) performing at least one assay for measuring or detecting a level of a biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in a sample obtained from the subject within about 24 hours after actual or suspected head injury, and (2) performing at least one head CT scan on the subject within a clinically relevant time frame, either simultaneously or sequentially (in any order); and (b) diagnosing the subject as likely to have TBI if the level of the biomarker is higher than a reference value and the head CT scan is negative for TBI. The sample can be a biological sample.

In yet another aspect, the disclosure relates to a method for aiding in determining whether a subject, such as a human subject, who may have, or is suspected of having, a head injury is likely to have a TBI. Specifically, the method includes performing an assay to measure or detect a level of a biomarker in a sample obtained from the subject within about 24 hours after an actual or suspected head injury, the biomarker level being ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample, the method includes diagnosing the subject as likely to have a traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and a head computed tomography (CT) scan performed on the subject within a clinically relevant time frame is negative for TBI or a head CT scan has not been performed on the subject. The sample can be a biological sample. In certain aspects, a head CT scan is performed on the subject. In other aspects, a head CT scan is not performed on the subject.

As described herein, when at least one assay and, optionally, at least one head CT scan are performed, the head CT scans can be performed simultaneously or sequentially in any order. When performed sequentially, the assay and the head CT scan can be performed within a clinically relevant time frame, e.g., within about 1 minute of each other, within about 2 minutes of each other, within about 3 minutes of each other, within about 4 minutes of each other, within about 5 minutes of each other, within about 10 minutes of each other, within about 15 minutes of each other, within about 20 minutes of each other, within about 25 minutes of each other, within about 30 minutes of each other, within about 45 minutes of each other, within about 50 minutes of each other, within about 60 minutes of each other, within about 1 hour of each other, within about 1.5 hours of each other, within about 2 hours of each other, within about 3 hours of each other, within about 4 hours of each other, within about 5 hours of each other, within about 6 hours of each other, within about 7 hours of each other, within about 8 hours of each other, within about 9 hours of each other, within about 10 hours of each other, within about 11 hours of each other, or within about 12 hours of each other.

In certain embodiments, the methods described herein can identify a subject who has experienced or may have experienced a head injury as having TBI based on one or more biomarker values if the subject has undergone a head CT scan that is negative for TBI. The methods described herein can identify subjects who have experienced TBI but may have been erroneously diagnosed as not having TBI based solely on the results of one or more head CT scans.

In certain embodiments, the method can include obtaining a sample within about 24 hours of the suspected injury in the subject and contacting the sample with an antibody to a biomarker for TBI, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, to form a complex between the antibody and the biomarker. The method can further include detecting the resulting antibody-biomarker complex.

In certain embodiments, the sample is collected from a subject, such as a human subject, within about 24 hours, e.g., within about 0 to about 6 hours, within about 0 to about 8 hours, within about 0 to about 10 hours, within about 0 to about 12 hours, within about 0 to about 18 hours, within about 6 hours to about 12 hours, within about 6 hours to about 18 hours, or within about 12 hours to about 18 hours, of a head injury (e.g., an actual injury) or suspected injury. For example, the sample can be collected from a subject, such as a human subject, within about 0 minutes, within about 30 minutes, within about 60 minutes, within about 90 minutes, within about 120 minutes, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours of the head injury or suspected injury. In certain embodiments, indications of the presence of a biomarker, such as UCH-L1, GFAP, or a combination thereof, appear within about 0 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 120 minutes, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after the head injury or suspected injury.

In general, a reference value of a biomarker, such as UCH-L1, GFAP, or a combination thereof, can be used as a benchmark for comparing and assessing results obtained by assaying test samples for UCH-L1. In general, in making such a comparison, a reference value of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained by performing a specific assay under suitable conditions and a sufficient number of times to correlate or associate the presence, amount, or concentration of the analyte with a particular stage or endpoint or a particular indicator of TBI. In general, a reference value of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained by assaying a reference subject (or subject population). The biomarker, such as UCH-L1, GFAP, or a combination thereof, that is measured can include fragments thereof, degradation products thereof, and/or enzymatic degradation products thereof.

In certain embodiments, the reference value for UCH-L1 is about 150 pg/mL to about 700 pg/mL, about 160 pg/mL to about 700 pg/mL, about 170 pg/mL to about 700 pg/mL, about 180 pg/mL to about 700 pg/mL, about 190 pg/mL to about 700 pg/mL, about 200 pg/mL to about 700 pg/mL, about 150 pg/mL to about 70 0 pg/mL, about 160 pg/mL to about 600 pg/mL, about 170 pg/mL to about 600 pg/mL, about 180 pg/mL to about 600 pg/mL, about 190 pg/mL to about 6 00 pg/mL, about 200 pg/mL to about 600 pg/mL, about 150 pg/mL to about 500 pg/mL, about 160 pg/mL to about 500 pg/mL, about 170 pg/mL to about 500 pg/mL, about 180 pg/mL to about 500 pg/mL, about 190 pg/mL to about 500 pg/mL, about 200 pg/mL to about 500 pg/mL, about 150 pg/mL ～about 400 pg/mL, about 160 pg/mL to about 400 pg/mL, about 170 pg/mL to about 400 pg/mL, about 180 pg/mL to about 400 pg/mL, about 190 pg/m L to about 400 pg/mL, about 200 pg/mL to about 400 pg/mL, about 150 pg/mL to about 300 pg/mL, about 160 pg/mL to about 300 pg/mL, about 170 pg/mL to about 300 pg/mL, about 180 pg/mL to about 300 pg/mL, about 190 pg/mL to about 300 pg/mL, or about 200 pg/mL to about 300 pg/mL.

Additionally or alternatively, in certain embodiments, the reference value for GFAP is about 30 pg/mL to about 1700 pg/mL, about 40 pg/mL to about 1700 pg/mL, about 50 pg/mL to about 1700 pg/mL, about 60 pg/mL to about 1700 pg/mL, about 70 pg/mL to about 1700 pg/mL, about 80 pg/mL to about 170 0 pg/mL, about 90 pg/mL to about 1700 pg/mL, about 100 pg/mL to about 1700 pg/mL, about 150 pg/mL to about 1700 pg/mL, about 200 pg /mL to about 1700 pg/mL, about 225 pg/mL to about 1700 pg/mL, about 250 pg/mL to about 1700 pg/mL, about 300 pg/mL to about 1700 pg/mL mL, about 400 pg/mL to about 1700 pg/mL, about 500 pg/mL to about 1700 pg/mL, about 600 pg/mL to about 1700 pg/mL, about 800 pg/mL ~ about 1700 pg/mL, about 900 pg/mL to about 1700 pg/mL, about 1000 pg/mL to about 1700 pg/mL, about 30 pg/mL to about 1600 pg/mL, About 40 pg/mL to about 1600 pg/mL, about 50 pg/mL to about 1600 pg/mL, about 60 pg/mL to about 1600 pg/mL, about 70 pg/mL to about 1600 p g/mL, about 80 pg/mL to about 1600 pg/mL, about 90 pg/mL to about 1600 pg/mL, about 100 pg/mL to about 1600 pg/mL, about 150 pg/mL ~ about 1600 pg/mL, about 200 pg/mL to about 1600 pg/mL, about 225 pg/mL to about 1600 pg/mL, about 250 pg/mL to about 1600 pg/mL, About 300 pg/mL to about 1600 pg/mL, about 400 pg/mL to about 1600 pg/mL, about 500 pg/mL to about 1600 pg/mL, about 600 pg/mL to about 1 600 pg/mL, about 800 pg/mL to about 1600 pg/mL, about 900 pg/mL to about 1600 pg/mL, about 1000 pg/mL to about 1600 pg/mL, about 3 0 pg/mL to about 1500 pg/mL, about 40 pg/mL to about 1500 pg/mL, about 50 pg/mL to about 1500 pg/mL, about 60 pg/mL to about 1500 pg/mL mL, about 70 pg/mL to about 1500 pg/mL, about 80 pg/mL to about 1500 pg/mL, about 90 pg/mL to about 1500 pg/mL, about 100 pg/mL to about 1 500 pg/mL, about 150 pg/mL to about 1500 pg/mL, about 200 pg/mL to about 1500 pg/mL, about 225 pg/mL to about 1500 pg/mL, about 25 0 pg/mL to about 1500 pg/mL, about 300 pg/mL to about 1500 pg/mL, about 400 pg/mL to about 1500 pg/mL, about 500 pg/mL to about 1500 pg/mL, about 600 pg/mL to about 1500 pg/mL, about 800 pg/mL to about 1500 pg/mL, about 900 pg/mL to about 1500 pg/mL, about 1000 p g/mL to about 1500 pg/mL, about 30 pg/mL to about 1400 pg/mL, about 40 pg/mL to about 1400 pg/mL, about 50 pg/mL to about 1400 pg/mL , about 60 pg/mL to about 1400 pg/mL, about 70 pg/mL to about 1400 pg/mL, about 80 pg/mL to about 1400 pg/mL, about 90 pg/mL to about 1400 pg/mL, about 100 pg/mL to about 1400 pg/mL, about 150 pg/mL to about 1400 pg/mL, about 200 pg/mL to about 1400 pg/mL, about 225 pg /mL to about 1400 pg/mL, about 250 pg/mL to about 1400 pg/mL, about 300 pg/mL to about 1400 pg/mL, about 400 pg/mL to about 1400 pg/mL mL, about 500 pg/mL to about 1400 pg/mL, about 600 pg/mL to about 1400 pg/mL, about 800 pg/mL to about 1400 pg/mL, about 900 pg/mL ～about 1400 pg/mL, about 1000 pg/mL to about 1400 pg/mL, about 30 pg/mL to about 1300 pg/mL, about 40 pg/mL to about 1300 pg/mL, about 50 pg/mL to about 1300 pg/mL, about 60 pg/mL to about 1300 pg/mL, about 70 pg/mL to about 1300 pg/mL, about 80 pg/mL to about 1300 pg /mL, about 90 pg/mL to about 1300 pg/mL, about 100 pg/mL to about 1300 pg/mL, about 150 pg/mL to about 1300 pg/mL, about 200 pg/mL - about 1300 pg/mL, about 225 pg/mL - about 1300 pg/mL, about 250 pg/mL - about 1300 pg/mL, about 300 pg/mL - about 1300 pg/mL, About 400 pg/mL to about 1300 pg/mL, about 500 pg/mL to about 1300 pg/mL, about 600 pg/mL to about 1300 pg/mL, about 800 pg/mL to about 1 300 pg/mL, about 900 pg/mL to about 1300 pg/mL, about 1000 pg/mL to about 1300 pg/mL, about 30 pg/mL to about 1200 pg/mL, about 40 pg/mL to about 1200 pg/mL, about 50 pg/mL to about 1200 pg/mL, about 60 pg/mL to about 1200 pg/mL, about 70 pg/mL to about 1200 pg/m L, about 80 pg/mL to about 1200 pg/mL, about 90 pg/mL to about 1200 pg/mL, about 100 pg/mL to about 1200 pg/mL, about 150 pg/mL to about 1 200 pg/mL, about 200 pg/mL to about 1200 pg/mL, about 225 pg/mL to about 1200 pg/mL, about 250 pg/mL to about 1200 pg/mL, about 30 0 pg/mL to about 1200 pg/mL, about 400 pg/mL to about 1200 pg/mL, about 500 pg/mL to about 1200 pg/mL, about 600 pg/mL to about 1200 pg/mL, about 800 pg/mL to about 1200 pg/mL, about 900 pg/mL to about 1200 pg/mL, or about 1000 pg/mL to about 1200 pg/mL.

In certain embodiments, the reference value for GFAP is from about 90 pg/mL to about 1680 pg/mL and the reference value for UCH-L1 is from about 220 pg/mL to about 670 pg/mL. In other embodiments, the reference value for GFAP is from about 110 pg/mL to about 950 pg/mL and the reference value for UCH-L1 is from about 160 pg/mL to about 320 pg/mL.

In certain embodiments, the method further includes administering traumatic brain injury treatment to a subject, such as a human subject, and/or monitoring the subject, as described below.

The type of assay utilized in the methods described herein is not critical and the test can be any assay known in the art, such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectrometric methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). The assays can also be utilized in clinical chemistry formats as known to those of ordinary skill in the art. Such assays are described in more detail in Sections 4-8 of this application. It is known in the art that values (e.g., calibrator and/or control reference values, cutoff values, thresholds, specificity, sensitivity, concentrations, etc.) used in assays utilizing a particular sample type (e.g., serum-based immunoassays or whole blood-based point-of-care devices) can be extrapolated to other assay formats by techniques known in the art, such as assay standardization. For example, one way assay normalization can be performed is by applying a factor to the calibrator utilized in the assay to raise or lower the sample concentration readings to obtain a slope consistent with the comparator method. Other methods for normalizing results from one assay to another are also well known and described in the literature (see, for example, David Wild, Immunoassay Handbook, 4th Edition, Chapter 3.5, pages 315-322, the disclosure of which is incorporated herein by reference).

3. Treatment and follow-up of subjects with head injury Subjects identified by the above methods can be treated or followed. In some embodiments, the method further comprises administering a traumatic brain injury treatment, such as any treatment known in the art, to a subject, such as a human subject. For example, traumatic brain injury treatment can take a variety of forms depending on the severity of the head injury. For example, in subjects with mild TBI, the treatment can include one or more of resting, refraining from physical activity such as exercise, avoiding light or wearing sunglasses when going out in bright places, taking medication to relieve headaches or migraines, taking antiemetic medication, etc. Treatment of patients with moderate, severe, or moderate-severe TBI may include administration of one or more appropriate medications (such as, for example, diuretics, anticonvulsants, medications to sedate the individual and place the individual in a drug-induced coma, or other pharmaceutical or biopharmaceutical drugs (known or developed in the future for the treatment of TBI)), one or more surgical procedures (such as, for example, evacuation of a hematoma, repair of a skull fracture, decompressive craniectomy, etc.), protection of the airway, and one or more therapies (such as, for example, one or more rehabilitation, cognitive behavioral therapy, anger management, counseling psychology, etc.). In certain embodiments, the method further includes following the subject, such as a human subject. In certain embodiments, the subject may be followed with CT scans or MRI techniques.

4. Methods for Measuring UCH-L1 Levels In the above methods, UCH-L1 levels can be measured by any means, including antibody-based methods such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, and chromatographic or spectrometric methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). The assays can also be utilized in clinical chemistry formats, as known to those of skill in the art.

In certain embodiments, measuring the UCH-L1 level includes contacting the sample with a first specific binding member and a second specific binding member. In certain embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In certain embodiments, measuring the UCH-L1 level includes contacting the sample with (1) a capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a complex of the capture antibody, UCH, and the L1 antigen (e.g., a complex of the UCH-L1 capture antibody and the UCH-L1 antigen), and (2) a detectable specific binding member such that a complex of the capture antibody, UCH-L1 antigen, and the detection antibody (e.g., a complex of the UCH-L1 capture antibody, UCH-L1 antigen, and the UCH-L1 detection antibody) is formed. The method includes contacting the sample simultaneously or sequentially in any order with a detection antibody (e.g., a UCH-L1 detection antibody) that contains a detectable label and binds to an epitope on UCH-L1 that is not bound to the capture antibody to form a complex of UCH-L1 antigen and detection antibody (e.g., a complex of UCH-L1 antigen and UCH-L1 detection antibody), and measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by the detectable label in the complex of the capture antibody, UCH-L1 antigen, and detection antibody.

In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a UCH-L1 antibody, as described below.

In certain embodiments, the sample is diluted or undiluted. The sample can be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In certain embodiments, the sample is about 1 to about 150 microliters or less, or about 1 to about 25 microliters or less.

Certain instruments other than point-of-care devices (e.g., the Abbott Laboratories ARCHITECT® instrument and other core laboratory instruments) appear to be capable of measuring UCH-L1 levels greater than 25,000 pg/mL in samples.

Other detection methods include, or can be adapted for use with, nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016/161402, the entire disclosure of which is incorporated herein by reference. Examples of nanowell devices are described in International Patent Publication No. WO 2016/161400, the entire disclosure of which is incorporated herein by reference.

5. UCH-L1 Antibodies The methods described herein can use an isolated antibody (referred to as a "UCH-L1 antibody") that specifically binds to ubiquitin carboxy-terminal hydrolase L1 ("UCH-L1") (or a fragment thereof). The UCH-L1 antibody can be used to assess UCH-L1 status as a measure of traumatic brain injury, to detect the presence of UCH-L1 in a sample, to quantitate the amount of UCH-L1 present in a sample, or to detect and quantitate the presence of UCH-L1 in a sample.

a. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1)
Ubiquitin carboxy-terminal hydrolase L1 ("UCH-L1"), also called "ubiquitin C-terminal hydrolase," is a deubiquitinating enzyme. UCH-L1 is a member of a gene family whose products hydrolyze small C-terminal adducts of ubiquitin to yield ubiquitin monomers. UCH-L1 expression is highly specific to neurons and cells of the diffuse endocrine system and their tumors. It is abundant in all neurons (representing 1-2% of total brain protein) and is specifically expressed in neurons and testis/ovary. The catalytic triad of UCH-L1 contains a cysteine at position 90, an aspartic acid at position 176, and a histidine at position 161, which are responsible for its hydrolase activity.

Human UCH-L1 can have the following amino acid sequence:

Human UCH-L1 may be a fragment or variant of SEQ ID NO:1. A fragment of UCH-L1 may be 5-225 amino acids long, 10-225 amino acids long, 50-225 amino acids long, 60-225 amino acids long, 65-225 amino acids long, 100-225 amino acids long, 150-225 amino acids long, 100-175 amino acids long, or 175-225 amino acids long. The fragment may comprise a consecutive number of amino acids from SEQ ID NO:1.

b. UCH-L1 recognizing antibody The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1, or a variant thereof. The antibody may be an anti-UCH-L1 antibody fragment or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody, or an antibody fragment such as a Fab fragment, or a mixture thereof. The antibody fragment or derivative may include F(ab') ₂ , Fv or scFv fragments. Antibody derivatives may be produced by peptidomimetics. Additionally, single chain antibodies may be produced by adapting techniques described for the production of single chain antibodies.

The anti-UCH-L1 antibody can be a chimeric anti-UCH-L1 antibody or a humanized anti-UCH-L1 antibody. In one embodiment, the humanized and chimeric antibodies are both monovalent. In one embodiment, the humanized and chimeric antibodies both comprise one Fab region linked to an Fc region.

Human antibodies can be obtained from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human in vivo immune response. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibodies can be the product of human and non-animal repertoires. Being of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, human anti-UCH-L1 antibodies can be selected and isolated using standard yeast display libraries and display techniques. For example, a library of naive human single chain variable fragments (scFv) can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

A humanized antibody is an antibody molecule derived from an antibody of a non-human species that binds to a desired antigen and can have one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

The antibody can be distinguished from known antibodies in that it has a biological function different from those known in the art.

(1) Epitope The antibody is capable of immunospecifically binding to UCH-L1 (SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibody is capable of immunospecifically recognizing and binding to at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids in the epitope region. The antibody is capable of immunospecifically recognizing and binding to at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids, or at least 10 consecutive amino acids in the epitope region.

c. Antibody Production/Production Antibodies can be produced by any of a variety of techniques, including techniques well known to those of skill in the art. In general, antibodies can be produced by cell culture techniques, including conventional techniques for producing monoclonal antibodies, or by transfecting the antibody genes, heavy and/or light chains into a suitable bacterial or mammalian cell host to produce the antibody, which may be recombinant. The various variations of the term "transfection" are intended to encompass a variety of techniques that are widely used to introduce foreign DNA into prokaryotic or eukaryotic host cells, including, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in both prokaryotic and eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells, with mammalian host cells being most preferred, since eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded, immunologically active antibodies.

Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) used with the DHFR selection marker, as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to permit expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. Of course, variations on the above procedure can be implemented. For example, it may be desirable to transfect the host cells with DNA encoding functional fragments of either the light and/or heavy chains of the antibody. Recombinant DNA techniques can be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies. Furthermore, bifunctional antibodies can be generated in which one heavy chain and one light chain are the first antibody (i.e., bind human UCH-L1) and the other heavy and light chains are specific for an antigen other than human UCH-L1, by crosslinking a first antibody with a second antibody by standard chemical crosslinking methods.

In a preferred recombinant expression system for an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate transfection. Inside the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLP promoter regulatory element, driving high levels of transcription of said genes. Because the recombinant expression vector also incorporates a DHFR gene, methotrexate selection/amplification can be used to select CHO cells transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to generate the recombinant expression vector, transfect the host cells, select the transformed cells, culture the host cells, and recover the antibody from the culture medium. Additionally, a method of synthesizing a recombinant antibody may be performed by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further include the step of isolating the recombinant antibody from the culture medium.

Methods for producing monoclonal antibodies include the creation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from splenocytes obtained from immunized animals. Animals can be immunized with UCH-L1 or fragments and/or variants thereof. The peptide used to immunize the animal can include amino acids encoding human Fc (e.g., a fragment crystallizable region or tail region of a human antibody). The splenocytes can then be immortalized, for example, by fusing with a myeloma cell fusion partner. Various fusion techniques can be utilized. For example, splenocytes and myeloma cells can be combined with a non-ionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine-aminopterin-thymidine (HAT) selection. Another technique involves electrofusion. After a sufficient time, usually about 1-2 weeks, hybrid colonies are observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. Various techniques can be used to further increase the yield, including injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be recovered from the ascites fluid or blood. Contaminants can be removed from the antibodies by conventional techniques such as chromatography, gel filtration, precipitation, and extraction. One example of a method that can be used in a process to purify antibodies is affinity chromatography.

The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) constitute covalent heterodimers, each of which contains an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab') ₂ fragment, which contains both antigen-binding sites.

Fv fragments can be generated by preferential proteolytic cleavage of IgM immunoglobulin molecules, and more rarely, IgG or IgA immunoglobulin molecules. Fv fragments can also be obtained using recombinant techniques. Fv fragments contain a non-covalent VH::VL heterodimer that contains an antigen-binding site that retains much of the antigen recognition and binding capacity of the native antibody molecule.

The antibody, antibody fragment, or derivative may include a set of heavy and light chain complementarity determining regions ("CDRs") and a set of heavy and light chain framework regions ("FRs") that flank the CDRs, support the CDRs, and define the spatial relationship between the CDRs. The CDR set may include three hypervariable regions of the heavy or light chain V region.

Other suitable methods of producing or isolating antibodies of the required specificity can also be used, including, but not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., non-limiting examples include bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, yeast, etc. display libraries) using methods known in the art, such as those available from a variety of vendors, such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK), and BioInvent (Lund, Sweden). See U.S. Patent Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, and 5,976,862. An alternative method relies on immunization of transgenic animals capable of producing a repertoire of human antibodies (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161), as known in the art and/or described herein. Such techniques include, but are not limited to, ribosome display technology (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody generation technology (e.g., lymphocyte-selected antibody method ("SLAM") (U.S. Pat. No. 5,627,052; Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA, 96:14130-14135); 93:7843-7848); gel microdrop flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).

Affinity matured antibodies can be generated by any one of a number of procedures known in the art. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol. , 154(7):3310-3319 (1995); Hawkins et al, J. Mol. Biol. , 226:889-896 (1992) describe random mutagenesis of CDR and/or framework residues. U.S. Patent No. 6,914,128 B1 describes selective mutations at selective mutagenesis positions and contact or hypermutation positions with activity enhancing amino acid residues.

Antibody variants can also be made by delivering a polynucleotide encoding the antibody to a suitable host to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such antibodies in its milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, and 5,304,489.

Antibody variants can also be made by delivering polynucleotides to provide variants in transgenic plants and cultured plant cells (e.g., tobacco, corn, and duckweed, for example, as non-limiting examples), specific parts, or cells cultured from said plant parts that produce such antibodies. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and references therein describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins using, for example, inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels that have biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and references therein. Antibody variants have been produced in large quantities from transgenic plant seeds containing antibody fragments such as single chain antibodies (scFv), as well as from tobacco seeds and potato tubers. See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and references therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

For example, antibody derivatives can be made by adding foreign sequences to alter immunogenicity or to reduce, enhance or alter binding affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable property. Generally, some or all of the non-human or human CDR sequences are maintained, and the non-human sequences in the variable and constant regions are replaced with human or other amino acids.

Small antibody fragments can be diabodies with two antigen-binding sites, which are fragments in which a heavy chain variable domain (VH) is linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). See, e.g., EP 404,097; WO 93/11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing of these two domains on the same antibody chain, these domains are paired with complementary domains on another antibody chain to form two antigen-binding sites. U.S. Patent No. 6,632,926 to Chen et al. discloses antibody mutants in which one or more amino acids are inserted into the hypervariable region of a parent antibody to provide a binding affinity for a target antigen that is at least about twice the binding affinity of the parent antibody for the antigen, the disclosure of which is incorporated herein by reference in its entirety.

The antibody can be a linear antibody. Procedures for making linear antibodies are known in the art and are described in Zapata et al., (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

The antibodies can be recovered and purified from the recombinant cell culture by known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

It may be useful to detectably label the antibody. Methods for conjugating antibodies to these substances are known in the art. For illustrative purposes only, antibodies can be labeled with a detectable moiety, such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled antibodies can be used in diagnostic techniques in vivo or in an isolated test sample. The antibody can be linked to a cytokine, a ligand, or another antibody. Substances suitable for coupling to antibodies to have an anti-tumor effect include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy including aluminum (III) phthalocyanine tetrasulfonic acid, hematoporphyrin, and phthalocyanine; iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m ( radionuclides such as 99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin A toxin, ricin A (deglycosylated ricin A and native ricin A), transforming growth factor-alpha toxin, Taiwan cobra (naja bacterial, plant, and other toxins such as cytotoxins derived from Aspergillus atra, and gelonin (a plant toxin); ribosome-inactivating proteins derived from plants, bacteria, and fungi such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein derived from Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleotide); liposomes encapsulating anticyst agents (e.g., antisense oligonucleotides, toxin-encoding plasmids, methotrexate, etc.); and other antibodies or antibody fragments such as F(ab).

Antibody production using hybridoma technology, lymphocyte-selected antibody technique (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

(1) Anti-UCH-L1 Monoclonal Antibody Using Hybridoma Technology Monoclonal antibodies can be produced using a variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or a combination thereof. For example, techniques known in the art and described in, for example, Harlow et al., Antibodies: A Laboratory Manual, second edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling, et al. Monoclonal antibodies can be made using hybridoma technology, including those taught in H. M., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is to be noted that the term "monoclonal antibody" as used herein is not limited to antibodies made by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is made.

The method for producing monoclonal antibodies and the antibodies produced by such a method may include the step of culturing hybridoma cells secreting the antibodies of the invention, preferably produced by fusing spleen cells isolated from an animal (e.g., rat or mouse) immunized with UCH-L1 with myeloma cells, followed by screening the hybridomas resulting from the fusion to select hybridoma clones secreting antibodies capable of binding to the polypeptides of the invention. Briefly, rats may be immunized with UCH-L1 antigen. In a preferred embodiment, the UCH-L1 antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). Such adjuvants may protect the polypeptide from rapid dispersion by isolating it in a localized depot, or may contain substances that stimulate the host to secrete chemotactic factors for macrophages and other components of the immune system. When administering a polypeptide, an immunization schedule in which the polypeptide is administered two or more times over a period of several weeks is preferred, although a single administration of the polypeptide may also be used.

After immunizing an animal with the UCH-L1 antigen, antibodies and/or antibody-producing cells can be obtained from the animal. Serum containing anti-UCH-L1 antibodies is obtained from the animal by bleeding or sacrificing the animal. The serum may be used as obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or anti-UCH-L1 antibodies may be purified from the serum. The serum or immunoglobulins obtained in this manner are polyclonal and therefore have a heterogeneous set of properties.

Once an immune response is detected (e.g., antibodies specific for the antigen UCH-L1 are detected in the rat serum), the rat spleen is removed and splenocytes are isolated. The splenocytes are then fused with any suitable myeloma cells (e.g., cells derived from cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US)) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding to UCH-L1. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing rats with positive hybridoma clones.

In another embodiment, immortalized hybridomas producing antibodies can be generated from the immunized animals. After immunization, the animals are sacrificed and splenic B cells are fused with immortalized myeloma cells as known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using UCH-L1 or a portion thereof, or cells expressing UCH-L1. In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA. An example of an ELISA screen is described in PCT Publication No. WO 00/37504.

Hybridomas producing anti-UCH-L1 antibodies are selected, cloned, and further screened for desirable properties, including robust hybridoma growth, high yield antibody production, and desirable antibody characteristics. Hybridomas can be cultured and propagated in vivo in syngeneic animals, animals that lack an immune system (e.g., nude mice), or in vitro in cell culture. Methods for selecting, cloning, and propagating hybridomas are well known to those of ordinary skill in the art.

In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow, or horse. In yet another preferred embodiment, the hybridoma is a human hybridoma formed by fusing a human cell expressing an anti-UCH-L1 antibody with a human non-secretory myeloma.

Antibody fragments that recognize non-specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F ₍ ab') ₂ fragments). The F(ab') ₂ fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule and contains both light chains (including the light chain variable region and the light chain constant region), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab') ₂ fragment is still capable of cross-linking antigen molecules in the same way as the parent IgG molecule.

(2) Anti-UCH-L1 Monoclonal Antibodies Using SLAM In another aspect of the invention, recombinant antibodies are produced from isolated single lymphocytes using a procedure known in the art as lymphocyte-selected antibody method (SLAM) and described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, an antigen-specific hemolytic plaque assay is used to screen for single cells (e.g., lymphocytes from any one of the immunized animals) that secrete an antibody of interest, and the antigen UCH-L1, a subunit of UCH-L1, or a fragment thereof is coupled to sheep red blood cells using a linker such as biotin and used to identify single cells that secrete antibodies with specificity for UCH-L1. After identifying cells secreting the antibody of interest, heavy and light chain variable region cDNAs can be rescued from the cells by reverse transcription PCR (RT-PCR) and then these variable regions can be expressed in the context of appropriate immunoglobulin constant regions (e.g., human constant regions) in mammalian host cells, such as COS or CHO cells. Immunoglobulin sequences from in vivo selected lymphocytes can be amplified and transfected into host cells and then further analyzed and selected in vitro, e.g., by panning the transfected cells to isolate cells expressing antibodies against UCH-L1. Amplified immunoglobulin sequences can be further manipulated in vitro, e.g., by in vitro affinity maturation techniques. See, e.g., PCT Publication Nos. WO 97/29131 and WO 00/56772.

(3) Anti-UCH-L1 Monoclonal Antibodies Using Transgenic Animals In another embodiment of the invention, antibodies are produced by immunizing a non-human animal containing part or all of the human immunoglobulin loci with a UCH-L1 antigen. In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an artificial mouse lineage that contains large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, and 6,130,364. See also PCT Publication Nos. WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560, and WO 00/37504. XENOMOUSE® transgenic mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human monoclonal antibodies. XENOMOUSE® transgenic mice contain approximately 80% of the human antibody repertoire through the introduction of megabase-sized germline conformation YAC fragments of the human heavy chain loci and x-light chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

(4) Anti-UCH-L1 Monoclonal Antibodies Using Recombinant Antibody Libraries In vitro methods for screening antibody libraries to identify antibodies with the desired UCH-L1 binding specificity can also be used to generate the antibodies of the present invention. Methods for such screening of recombinant antibody libraries are well known in the art and are described, for example, in U.S. Pat. No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92/18619 (Kang et al.); PCT Publication No. WO 91/17271 (Dower et al.); PCT Publication No. WO 92/20791 (Winter et al.); PCT Publication No. WO 92/15679 (Markland et al.); PCT Publication No. WO 93/01288 (Breitling et al.); PCT Publication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO 92/09690 (Garrard et al.), the disclosures of each of which are incorporated herein by reference. al. ); Fuchs et al. , Bio/Technology, 9:1369-1372 (1991); Hay et al. , Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al. , Science, 246:1275-1281 (1989); McCafferty et al. , Nature, 348:552-554 (1990); Griffiths et al. , EMBO J. , 12:725-734 (1993); Hawkins et al. , J. Mol. Biol. , 226:889-896 (1992); Clackson et al. , Nature, 352:624-628 (1991); Gram et al. , Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al. , Bio/Technology, 9:1373-1377 (1991); Hoogenboom et al. , Nucl. Acids Res. , 19:4133-4137 (1991); Barbas et al. , Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003/0186374; and PCT Publication No. WO 97/29131.

The recombinant antibody library can be derived from a subject immunized with UCH-L1 or a portion of UCH-L1. Alternatively, the recombinant antibody library can be derived from a naive subject (i.e., a subject not immunized with UCH-L1), such as a human antibody library derived from a human subject not immunized with human UCH-L1. The antibodies of the invention are selected by screening the recombinant antibody library with a peptide comprising human UCH-L1 to select for antibodies that recognize UCH-L1. Methods for performing such screening and selection are well known in the art, as described in the references listed in the previous paragraph. To select an antibody of the invention having a particular binding affinity for UCH-L1 (e.g., an antibody that dissociates from human UCH-L1 with a particular K _off rate constant), surface plasmon resonance methods known in the art can be used to select an antibody with the desired K _off rate constant. To select an antibody of the invention having a particular neutralizing activity against hUCH-L1 (e.g., an antibody with a particular IC ₅₀ ), standard methods known in the art for assessing inhibition of UCH-L1 activity can be used.

In one aspect, the invention relates to an isolated antibody or antigen-binding portion thereof that binds to human UCH-L1. The antibody is preferably a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies can be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that contain polynucleotide sequences encoding said domains. Such phage can be used to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). For example, labeled antigen or antigen bound or captured to a solid surface or bead can be used to antigen-select or identify phage that express an antigen binding domain that binds to the antigen of interest. Phages used in these methods are generally filamentous phage containing fd and M13 binding domains expressed from phages recombinantly fused to phage gene III or gene VIII proteins with Fab antibody domains, Fv antibody domains, or disulfide bond stabilized Fv antibody domains. Examples of phage display methods that can be used to generate antibodies include those described in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al. , J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al. , Eur. J. Immunol. , 24:952-958 (1994); Persic et al. , Gene, 187:9-18 (1997); Burton et al. ,Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92/01047; PCT Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and U.S. Pat. Nos. 5,698,426, 5,2 Examples of the methods disclosed in the above-mentioned patents include those disclosed in Nos. 23,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

After phage selection, as described in the above references, antibody encoding regions from the phage can be isolated and used to generate full length antibodies, including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in more detail below (see, e.g., PCT Publication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., J. Immunol., 34:26-34 (1995)). Techniques are also available for recombinant production of Fab, Fab', and F(ab') ₂ fragments using methods known in the art, such as those disclosed in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., J. Immunol. 1999, 11:1112-1113 (1994). Examples of techniques that can be used to make single chain Fv and antibodies include those disclosed in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., J. Immunol. 1999, 11:1112-1113 (1994). , Science, 240:1038-1041 (1988).

As an alternative to screening recombinant antibody libraries using phage display techniques, other methods known in the art for screening large combinatorial libraries can also be applied to identify antibodies of the invention. One example of an alternative expression system is the expression of recombinant antibody libraries as RNA-protein fusions, as described in PCT Publication No. WO 98/31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, in vitro translation of synthetic mRNAs with the peptidyl acceptor antibiotic puromycin attached to their 3' ends creates covalent fusions of the mRNAs and the peptides or proteins they encode. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein (e.g., an antibody or portion thereof) (e.g., binding of the antibody or portion thereof to a dual-specific antigen). Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above and further subjected to affinity maturation by screening additional rounds of mRNA-peptide fusions that introduce mutations into the originally selected sequence, as described above, or by other in vitro affinity maturation methods of recombinant antibodies. A preferred example of this method is the PROfusion display technology.

In another approach, antibodies can be made using yeast display methods known in the art, in which antibody domains are tethered to the yeast cell wall using genetic engineering techniques and displayed on the surface of the yeast. Such yeast can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or mouse). Examples of yeast display methods that can be used to make antibodies include those disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

d. Production of Recombinant UCH-L1 Antibodies Antibodies can be produced by any of a number of techniques known in the art. For example, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques and expressed from the host cell. Variations of the term "transfection" are intended to encompass a variety of techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, including, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the invention can be expressed in either prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, with mammalian host cells being most preferred, since eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete a correctly folded, immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) used with the DHFR selection marker, as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a time sufficient to allow expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. Of course, variations on the above procedures can be implemented. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and/or heavy chains of the antibodies of the invention. Recombinant DNA techniques can be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the invention. Furthermore, bifunctional antibodies can be generated in which one heavy and light chain is an antibody of the invention (i.e., binds human UCH-L1) and the other heavy and light chains are specific for an antigen other than human UCH-L1, by crosslinking the antibodies of the invention with a second antibody by standard chemical crosslinking methods.

In a preferred recombinant expression system for the antibody or antigen-binding portion thereof of the present invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate transfection. Inside the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLP promoter regulatory element, driving high levels of transcription of said genes. Because the recombinant expression vector also incorporates a DHFR gene, methotrexate selection/amplification can be used to select CHO cells transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to generate the recombinant expression vector, transfect the host cells, select the transformed cells, culture the host cells, and recover the antibody from the culture medium. The present invention further provides a method for synthesizing a recombinant antibody of the present invention by culturing the host cells of the present invention in an appropriate culture medium until a recombinant antibody of the present invention is synthesized. The method may further include isolating the recombinant antibody from the culture medium.

(1) Humanized Antibodies A humanized antibody can be an antibody, or a variant, derivative, analog or portion thereof, that immunospecifically binds to an antigen of interest and contains a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody can be derived from a non-human species antibody that binds a desired antigen and has one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

The term "substantially" as used herein with respect to CDRs refers to a CDR having an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') ₂ , FabC, Fv), in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are framework regions of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. In some embodiments, the humanized antibody comprises both a light chain and at least the variable domain of a heavy chain. The antibody further comprises the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only comprises a humanized light chain. In some embodiments, a humanized antibody only comprises a humanized heavy chain. In certain embodiments, a humanized antibody only comprises humanized variable domains of the light and/or heavy chain.

Humanized antibodies can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can contain sequences from more than one class or isotype, and specific constant domains can be selected to optimize desired effector functions using techniques well known in the art.

The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in one embodiment, such mutations will not be extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of cognate immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.

Immune responses to rodent anti-human antibodies limit the duration and effectiveness of therapeutic applications of these moieties in human recipients, and humanized antibodies can be designed to minimize such undesirable immune responses. Humanized antibodies can have one or more amino acid residues introduced from a non-human source. These non-human residues are often referred to as "import" residues and are typically taken from the variable domain. Humanization can be performed by substituting hypervariable region sequences for corresponding sequences in a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. See, for example, U.S. Pat. No. 4,816,567, the disclosure of which is incorporated herein by reference. Humanized antibodies can be human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies. Humanization or engineering of the antibodies of the present invention can be carried out using any known method, including, but not limited to, those described in U.S. Patent Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567.

Humanized antibodies can maintain high affinity for UCH-L1 and other favorable biological properties. Humanized antibodies can be generated by analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are widely available. Computer programs are available that draw and display predicted three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the predicted role of the residues in the function of the candidate immunoglobulin sequence, i.e., residues that influence the ability of the candidate immunoglobulin to bind its antigen. FR residues can thus be selected and combined from the recipient and import sequences to obtain desired antibody characteristics, such as increased affinity for UCH-L1. In general, it appears that the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, human antibodies can be isolated from libraries by PROfusion and/or yeast-related technologies. Transgenic animals (e.g., mice capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization) can also be produced. For example, homozygous deletion of the antibody heavy-chain joining region ( _JH ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germ-line immunoglobulin gene sequences into such germ-line mutant mice will result in the production of human antibodies after antigen challenge. Humanized or fully human antibodies can be made according to the methods described in U.S. Pat. Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,017,517, 6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690, 6,682,928, and 6,984,720, the disclosures of each of which are incorporated herein by reference.

e. Anti-UCH-L1 Antibody Anti-UCH-L1 antibodies can be produced using the techniques described above and other conventional techniques known in the art. In certain embodiments, anti-UCH-L1 antibodies are available from United State Biological (catalog number: 031320), Cell Signaling Technology (catalog number: 3524), Sigma-Aldrich (catalog number: HPA005993), Santa Cruz Biotechnology, Inc. (catalog number: sc-58593 or sc-58594), R&D Systems (catalog number: MAB6007), Novus Biologicals (catalog number: NB600-1160), Biorbyte (catalog number: orb33715), Enzo Life Sciences, Inc. (catalog number: ADI-905-520-1), Bio-Rad (catalog number: VMA00004), BioVision (catalog number: 6130-50), Abcam (catalog number: ab75275 or ab104938), Invitrogen Antibodies (catalog number: 480012), ThermoFisher Scientific (catalog number: MA1-46079, MA5-17235, MA1-90008, or MA1-83428), EMD Millipore (catalog number: MABN48), or Sino Biological Inc. The antibody can be an unconjugated anti-UCH-L1 antibody, such as the UCH-L1 antibody available from BioVision (Cat. No. 50690-R011). The UCH-L1 antibody can be conjugated to a fluorophore, such as the conjugated UCH-L1 antibody available from BioVision (Cat. No. 6960-25) or Aviva Systems Biology (Cat. Nos. OAAF01904-FITC).

6. Methods for Measuring GFAP Levels In the above methods, GFAP levels can be measured by any means, including antibody-based methods such as immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, and chromatographic or spectrometric methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS). The assays can also be utilized in clinical chemistry formats, as known to those skilled in the art.

In certain embodiments, measuring the GFAP level comprises contacting the sample with a first specific binding member and a second specific binding member. In certain embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In certain embodiments, measuring the GFAP level includes contacting the sample with (1) a capture antibody (e.g., a GFAP capture antibody) that binds to an epitope on GFAP or a GFAP fragment to form a complex of the capture antibody and the GFAP antigen (e.g., a complex of the GFAP capture antibody and the GFAP antigen), and (2) a detection antibody (e.g., a GFAP detection antibody) that contains a detectable label and binds to an epitope on GFAP that is not bound to the capture antibody to form a complex of the GFAP antigen and the detection antibody (e.g., a complex of the GFAP antigen and the GFAP detection antibody), simultaneously or sequentially in any order, so that a complex of the capture antibody, the GFAP antigen, and the detection antibody (e.g., a complex of the GFAP capture antibody, the GFAP antigen, and the GFAP detection antibody) is formed, and measuring the amount or concentration of GFAP in the sample based on a signal generated by the detectable label in the complex of the capture antibody, the GFAP antigen, and the detection antibody.

In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a GFAP antibody, as described below.

In certain embodiments, the sample is diluted or undiluted. The sample can be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In certain embodiments, the sample is about 1 to about 150 microliters or less, or about 1 to about 25 microliters or less.

Certain instruments other than point-of-care devices (e.g., the Abbott Laboratories instrument ARCHITECT® and other core laboratory instruments) appear to be capable of measuring GFAP levels in samples greater than 25,000 pg/mL.

Other detection methods include, or can be adapted for use with, nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016/161402, the entire disclosure of which is incorporated herein by reference. Examples of nanowell devices are described in International Patent Publication No. WO 2016/161400, the entire disclosure of which is incorporated herein by reference.

7. GFAP Antibodies The methods described herein can use isolated antibodies (referred to as "GFAP antibodies") that specifically bind to glial fibrillary acidic protein ("GFAP") (or fragments thereof). The GFAP antibodies can be used to assess GFAP status as a measure of traumatic brain injury, to detect the presence of GFAP in a sample, to quantitate the amount of GFAP present in a sample, or to detect and quantitate the presence of GFAP in a sample.

a. Glial fibrillary acidic protein (GFAP)
Glial fibrillary acidic protein (GFAP) is a 50 kDa cytoplasmic fibrillar protein that constitutes part of the cytoskeleton in astrocytes and has proven to be the most specific marker for astrocyte-derived cells. GFAP protein is encoded by the GFAP gene in humans. GFAP is the major intermediate filament of mature astrocytes. In the central rod domain of the molecule, GFAP has significant structural homology with other intermediate filaments. GFAP is involved in astrocyte motility and shape by providing structural stability to astrocyte processes. Glial fibrillary acidic protein and its degradation products (GFAP-BDPs) are brain-specific proteins that are released into the blood as part of the pathophysiological response after traumatic brain injury (TBI). After injury of the human CNS caused by trauma, genetic insults, or chemicals, astrocytes proliferate and show extensive hypertrophy of cell bodies and processes, and GFAP is significantly upregulated. On the other hand, a progressive decrease in GFAP production occurs with increasing astrocyte malignancy. GFAP can also be detected in Schwann cells, intestinal glial cells, salivary gland neoplasms, metastatic renal carcinoma, epiglottic cartilage, pituitary cells, immature oligodendrocytes, papillary meningiomas, and breast myoepithelial cells.

Human GFAP can have the following amino acid sequence:

The human GFAP may be a fragment or variant of SEQ ID NO:2. The GFAP fragment may be 5-400 amino acids long, 10-400 amino acids long, 50-400 amino acids long, 60-400 amino acids long, 65-400 amino acids long, 100-400 amino acids long, 150-400 amino acids long, 100-300 amino acids long, or 200-300 amino acids long. The fragment may include a consecutive number of amino acids derived from SEQ ID NO:2. The human GFAP fragment or variant of SEQ ID NO:2 may be a GFAP degradation product (BDP). The GFAP BDP may be 38 kDa, 42 kDa (low molecular weight 41 kDa), 47 kDa (low molecular weight 45 kDa), 25 kDa (low molecular weight 23 kDa), 19 kDa, or 20 kDa.

b. GFAP-Recognizing Antibody The antibody is an antibody that binds to GFAP, a fragment thereof, an epitope of GFAP, or a variant thereof. The antibody may be an anti-GFAP antibody fragment or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single chain antibody, an affinity matured antibody, a human antibody, a humanized antibody, a fully human antibody, or an antibody fragment such as a Fab fragment, or a mixture thereof. The antibody fragment or derivative may include F(ab') ₂ , Fv or scFv fragments. Antibody derivatives may be produced by peptidomimetics. Additionally, single chain antibodies may be produced by adapting techniques described for the production of single chain antibodies.

The anti-GFAP antibody can be a chimeric anti-GFAP antibody or a humanized anti-GFAP antibody. In one embodiment, the humanized and chimeric antibodies are both monovalent. In one embodiment, the humanized and chimeric antibodies both comprise one Fab region linked to an Fc region.

Human antibodies can be obtained from phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human in vivo immune response. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibodies can be the product of human and non-animal repertoires. Being of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, human anti-GFAP antibodies can be selected and isolated using standard yeast display libraries and display techniques. For example, a library of naive human single chain variable fragments (scFv) can be used to select human anti-GFAP antibodies. Transgenic animals can be used to express human antibodies.

A humanized antibody is an antibody molecule derived from an antibody of a non-human species that binds to a desired antigen and can have one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

The antibody can be distinguished from known antibodies in that it has a biological function different from those known in the art.

(1) Epitope The antibody can immunospecifically bind to GFAP (SEQ ID NO: 2), a fragment thereof, or a variant thereof. The antibody can immunospecifically recognize and bind to at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids in the epitope region. The antibody can immunospecifically recognize and bind to at least 3 consecutive amino acids, at least 4 consecutive amino acids, at least 5 consecutive amino acids, at least 6 consecutive amino acids, at least 7 consecutive amino acids, at least 8 consecutive amino acids, at least 9 consecutive amino acids, or at least 10 consecutive amino acids in the epitope region.

c. Antibody Production/Production Antibodies can be produced by any of a variety of techniques, including techniques well known to those of skill in the art. In general, antibodies can be produced by cell culture techniques, including conventional techniques for producing monoclonal antibodies, or by transfecting the antibody genes, heavy and/or light chains into a suitable bacterial or mammalian cell host to produce the antibody, which may be recombinant. The various variations of the term "transfection" are intended to encompass a variety of techniques that are widely used to introduce foreign DNA into prokaryotic or eukaryotic host cells, including, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although antibodies can be expressed in both prokaryotic and eukaryotic host cells, it is preferred to express antibodies in eukaryotic cells, with mammalian host cells being most preferred, since eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded, immunologically active antibodies.

Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO cells) used with the DHFR selection marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a time sufficient to allow expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. Of course, variations on the above procedures can be implemented. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and/or heavy chains of the antibody. Recombinant DNA techniques can be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies. Furthermore, bifunctional antibodies can be generated in which one heavy chain and one light chain are the first antibody (i.e., bind human GFAP) and the other heavy and light chains are specific for an antigen other than human GFAP, by crosslinking a first antibody with a second antibody by standard chemical crosslinking methods.

In a preferred recombinant expression system for an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate transfection. Inside the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLP promoter regulatory element, driving high levels of transcription of said genes. Because the recombinant expression vector also incorporates a DHFR gene, methotrexate selection/amplification can be used to select CHO cells transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to generate the recombinant expression vector, transfect the host cells, select the transformed cells, culture the host cells, and recover the antibody from the culture medium. Additionally, a method of synthesizing a recombinant antibody may be performed by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further include the step of isolating the recombinant antibody from the culture medium.

Methods for producing monoclonal antibodies include the creation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from splenocytes obtained from immunized animals. The animals can be immunized with GFAP or fragments and/or variants thereof. The peptide used to immunize the animals can include amino acids encoding human Fc (e.g., a fragment crystallizable region or tail region of a human antibody). The splenocytes can then be immortalized, for example, by fusing with a myeloma cell fusion partner. Various fusion techniques can be used. For example, splenocytes and myeloma cells can be combined with a non-ionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine-aminopterin-thymidine (HAT) selection. Another technique involves electrofusion. After a sufficient time, usually about 1-2 weeks, hybrid colonies are observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. Various techniques can be used to further increase the yield, including injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be recovered from the ascites fluid or blood. Contaminants can be removed from the antibodies by conventional techniques such as chromatography, gel filtration, precipitation, and extraction. One example of a method that can be used in a process to purify antibodies is affinity chromatography.

The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) constitute covalent heterodimers that each contain an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab') ₂ fragment, which contains both antigen-binding sites.

Fv fragments can be generated by preferential proteolytic cleavage of IgM immunoglobulin molecules, and more rarely, IgG or IgA immunoglobulin molecules. Fv fragments can also be obtained using recombinant techniques. Fv fragments contain a non-covalent VH::VL heterodimer that contains an antigen-binding site that retains much of the antigen recognition and binding capacity of the native antibody molecule.

The antibody, antibody fragment, or derivative may include a set of heavy and light chain complementarity determining regions ("CDRs") and a set of heavy and light chain frameworks ("FRs") that flank the CDRs, support the CDRs, and define the spatial relationship between the CDRs. The CDR set may include three hypervariable regions of the heavy or light chain V region.

Other suitable methods of producing or isolating antibodies of the required specificity can also be used, including, but not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., non-limiting examples include bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, yeast, etc. display libraries) using methods known in the art, such as those available from a variety of vendors, such as Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg, Del.), Biovation (Aberdeen, Scotland, UK), and BioInvent (Lund, Sweden). See U.S. Patent Nos. 4,704,692, 5,723,323, 5,763,192, 5,814,476, 5,817,483, 5,824,514, and 5,976,862. An alternative method relies on immunization of transgenic animals capable of producing a repertoire of human antibodies (e.g., SCID mice, Nguyen et al. (1997) Microbiol. Immunol. 41:901-907; Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161), as known in the art and/or described herein. Such techniques include, but are not limited to, ribosome display technology (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibody generation technology (e.g., lymphocyte-selected antibody method ("SLAM") (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996) Proc. Natl. Acad. Sci. USA, 96:14130-14135); 93:7843-7848); gel microdrop flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al. (1995) J. Imm. Meth. 182:155-163; Kenny et al. (1995) Bio/Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134 (1994)).

Affinity matured antibodies can be generated by any one of a number of procedures known in the art. For example, Marks et al., BioTechnology, 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol. , 154(7):3310-3319 (1995); Hawkins et al, J. Mol. Biol. , 226:889-896 (1992) describe random mutagenesis of CDR and/or framework residues. U.S. Patent No. 6,914,128 B1 describes selective mutations at selective mutagenesis positions and contact or hypermutation positions with activity enhancing amino acid residues.

Antibody variants can also be made by delivering a polynucleotide encoding the antibody to a suitable host to produce a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., that produces such antibodies in its milk. These methods are known in the art and are described, for example, in U.S. Patent Nos. 5,827,690, 5,849,992, 4,873,316, 5,849,992, 5,994,616, 5,565,362, and 5,304,489.

Antibody variants can also be made by delivering polynucleotides to provide variants in transgenic plants and cultured plant cells (e.g., tobacco, corn, and duckweed, for example, as non-limiting examples), specific parts, or cells cultured from said plant parts that produce such antibodies. For example, Cramer et al. (1999) Curr. Top. Microbiol. Immunol. 240:95-118 and references therein describe the production of transgenic tobacco leaves that express large amounts of recombinant proteins using, for example, inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels that have biological activity equivalent to mammalian proteins produced in other recombinant systems or purified from natural sources. See, for example, Hood et al., Adv. Exp. Med. Biol. (1999) 464:127-147 and references therein. Antibody variants have been produced in large quantities from transgenic plant seeds containing antibody fragments such as single chain antibodies (scFv), as well as from tobacco seeds and potato tubers. See, for example, Conrad et al. (1998) Plant Mol. Biol. 38:101-109 and references therein. Thus, antibodies can also be produced using transgenic plants according to known methods.

For example, antibody derivatives can be made by adding foreign sequences to alter immunogenicity or to reduce, enhance or alter binding affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable property. Generally, some or all of the non-human or human CDR sequences are maintained, and the non-human sequences in the variable and constant regions are replaced with human or other amino acids.

Small antibody fragments can be diabodies with two antigen-binding sites, which are fragments in which a heavy chain variable domain (VH) is linked to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). See, e.g., EP 404,097; WO 93/11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too short to allow pairing of these two domains on the same antibody chain, these domains are paired with complementary domains on another antibody chain to form two antigen-binding sites. U.S. Patent No. 6,632,926 to Chen et al. discloses antibody mutants in which one or more amino acids are inserted into the hypervariable region of a parent antibody to provide a binding affinity for a target antigen that is at least about twice the binding affinity of the parent antibody for the antigen, the disclosure of which is incorporated herein by reference in its entirety.

The antibody can be a linear antibody. Procedures for making linear antibodies are known in the art and are described in Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

The antibodies can be recovered and purified from the recombinant cell culture by known methods, including, but not limited to, Protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

It may be useful to detectably label the antibody. Methods for conjugating antibodies to these substances are known in the art. For illustrative purposes only, antibodies can be labeled with a detectable moiety, such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled antibodies can be used in diagnostic techniques in vivo or in an isolated test sample. The antibody can be linked to a cytokine, a ligand, or another antibody. Substances suitable for coupling to antibodies to have an anti-tumor effect include cytokines such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for photodynamic therapy including aluminum (III) phthalocyanine tetrasulfonic acid, hematoporphyrin, and phthalocyanine; iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m ( radionuclides such as 99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin A toxin, ricin A (deglycosylated ricin A and native ricin A), transforming growth factor-alpha toxin, Taiwan cobra (naja bacterial, plant, and other toxins such as cytotoxins derived from Aspergillus atra, and gelonin (a plant toxin); ribosome-inactivating proteins derived from plants, bacteria, and fungi such as restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (a ribosome-inactivating protein derived from Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleotide); liposomes encapsulating anticyst agents (e.g., antisense oligonucleotides, toxin-encoding plasmids, methotrexate, etc.); and other antibodies or antibody fragments such as F(ab).

Antibody production using hybridoma technology, lymphocyte-selected antibody technique (SLAM), transgenic animals, and recombinant antibody libraries is described in more detail below.

(1) Anti-GFAP monoclonal antibodies using hybridoma technology Monoclonal antibodies can be produced using a variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or a combination thereof. For example, the techniques known in the art and described in, for example, Harlow et al., Antibodies: A Laboratory Manual, second edition, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988); Hammerling, et al. Monoclonal antibodies can be made using hybridoma technology, including those taught in H. M., In Monoclonal Antibodies and T-Cell Hybridomas, (Elsevier, N.Y., 1981). It is to be noted that the term "monoclonal antibody" as used herein is not limited to antibodies made by hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is made.

The method for producing monoclonal antibodies and the antibodies produced by such a method may include a step of culturing hybridoma cells secreting the antibodies of the invention, preferably produced by fusing spleen cells isolated from an animal (e.g., rat or mouse) immunized with GFAP with myeloma cells, followed by screening the hybridomas resulting from the fusion and selecting hybridoma clones secreting antibodies capable of binding to the polypeptides of the invention. In summary, rats may be immunized with GFAP antigen. In a preferred embodiment, the GFAP antigen is administered with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). Such adjuvants may protect the polypeptide from rapid dispersion by isolating it in a localized depot, or may contain substances that stimulate the host to secrete chemotactic factors for macrophages and other components of the immune system. When administering a polypeptide, an immunization schedule in which the polypeptide is administered two or more times over a period of several weeks is preferred, although a single administration of the polypeptide may also be used.

After immunizing an animal with the GFAP antigen, antibodies and/or antibody-producing cells can be obtained from the animal. Serum containing anti-GFAP antibodies is obtained from the animal by bleeding or sacrificing the animal. The serum may be used as obtained from the animal, an immunoglobulin fraction may be obtained from the serum, or the anti-GFAP antibodies may be purified from the serum. The serum or immunoglobulins obtained in this manner are polyclonal and therefore have a heterogeneous set of properties.

Once an immune response is detected (e.g., antibodies specific for the antigen GFAP are detected in the rat serum), the rat spleen is removed and splenocytes are isolated. The splenocytes are then fused with any suitable myeloma cells (e.g., cells derived from cell line SP20 available from the American Type Culture Collection (ATCC, Manassas, Va., US)) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. Hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding to GFAP. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing rats with positive hybridoma clones.

In another embodiment, antibody producing immortal hybridomas can be made from immunized animals. After immunization, the animals are sacrificed and splenic B cells are fused with immortal myeloma cells as known in the art. See, e.g., Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secretory cell lines). After fusion and antibiotic selection, the hybridomas are screened using GFAP or a portion thereof, or cells expressing GFAP. In a preferred embodiment, initial screening is performed using an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA), preferably ELISA. An example of an ELISA screen is described in PCT Publication No. WO 00/37504.

Hybridomas producing anti-GFAP antibodies are selected, cloned, and further screened for desirable properties, including robust hybridoma growth, high yields of antibody production, and desirable antibody characteristics. Hybridomas can be cultured and expanded in vivo in syngeneic animals, animals that lack an immune system (e.g., nude mice), or in vitro in cell culture. Methods for selecting, cloning, and expanding hybridomas are well known to those of ordinary skill in the art.

In a preferred embodiment, the hybridoma is a rat hybridoma. In another embodiment, the hybridoma is produced in a non-human, non-rat species, such as mouse, sheep, pig, goat, cow, or horse. In yet another preferred embodiment, the hybridoma is a human hybridoma formed by fusing a human cell expressing an anti-GFAP antibody with a human non-secretory myeloma.

Antibody fragments that recognize non-specific epitopes can be produced by known techniques. For example, Fab and F(ab')2 fragments of the invention can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce two identical Fab fragments) or pepsin (to produce F ₍ ab') ₂ fragments). The F(ab') ₂ fragment of an IgG molecule retains the two antigen-binding sites of the larger ("parent") IgG molecule and contains both light chains (including the light chain variable region and the light chain constant region), the CH1 domain of the heavy chain, and the disulfide-forming hinge region of the parent IgG molecule. Thus, the F(ab') ₂ fragment is still capable of cross-linking antigen molecules in the same way as the parent IgG molecule.

(2) Anti-GFAP Monoclonal Antibodies Using SLAM In another aspect of the invention, recombinant antibodies are produced from isolated single lymphocytes using a procedure known in the art as lymphocyte-selected antibody method (SLAM) and described in U.S. Patent No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this method, an antigen-specific hemolytic plaque assay is used to screen for single cells (e.g., lymphocytes from any one of the immunized animals) that secrete an antibody of interest, and the antigen GFAP, a subunit of GFAP, or a fragment thereof is coupled to sheep red blood cells using a linker such as biotin and used to identify single cells that secrete antibodies with specificity for GFAP. After identifying cells that secrete the antibody of interest, heavy and light chain variable region cDNAs can be rescued from the cells by reverse transcription PCR (RT-PCR) and then these variable regions can be expressed in the context of appropriate immunoglobulin constant regions (e.g., human constant regions) in mammalian host cells, such as COS or CHO cells. Immunoglobulin sequences from in vivo selected lymphocytes can be amplified and transfected into host cells and then further analyzed and selected in vitro, e.g., by panning the transfected cells to isolate cells that express antibodies against GFAP. Amplified immunoglobulin sequences can be further engineered in vitro, e.g., by in vitro affinity maturation techniques. See, e.g., PCT Publication Nos. WO 97/29131 and WO 00/56772.

(3) Anti-GFAP Monoclonal Antibodies Using Transgenic Animals In another embodiment of the invention, antibodies are produced by immunizing a non-human animal containing part or all of the human immunoglobulin loci with a GFAP antigen. In one embodiment, the non-human animal is a XENOMOUSE® transgenic mouse, an artificial mouse lineage that contains large fragments of the human immunoglobulin loci and is deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 (1994) and U.S. Patent Nos. 5,916,771, 5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, and 6,130,364. See also PCT Publication Nos. WO 91/10741, WO 94/02602, WO 96/34096, WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO 99/53049, WO 00/09560, and WO 00/37504. XENOMOUSE® transgenic mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human monoclonal antibodies. XENOMOUSE® transgenic mice contain approximately 80% of the human antibody repertoire through the introduction of megabase-sized germline conformation YAC fragments of the human heavy chain loci and x-light chain loci. See Mendez et al., Nature Genetics, 15:146-156 (1997); Green and Jakobovits, J. Exp. Med., 188:483-495 (1998), the disclosures of which are incorporated herein by reference.

(4) Anti-GFAP Monoclonal Antibodies Using Recombinant Antibody Libraries In vitro methods for screening antibody libraries to identify antibodies with the desired GFAP-binding specificity can also be used to generate the antibodies of the present invention. Methods for such screening of recombinant antibody libraries are well known in the art and are described, for example, in U.S. Pat. No. 5,223,409 (Ladner et al.); PCT Publication No. WO 92/18619 (Kang et al.); PCT Publication No. WO 91/17271 (Dower et al.); PCT Publication No. WO 92/20791 (Winter et al.); PCT Publication No. WO 92/15679 (Markland et al.); PCT Publication No. WO 93/01288 (Breitling et al.); PCT Publication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO 92/09690 (Garrard et al.), the disclosures of each of which are incorporated herein by reference. al. ); Fuchs et al. , Bio/Technology, 9:1369-1372 (1991); Hay et al. , Hum. Antibod. Hybridomas, 3:81-85 (1992); Huse et al. , Science, 246:1275-1281 (1989); McCafferty et al. , Nature, 348:552-554 (1990); Griffiths et al. , EMBO J. , 12:725-734 (1993); Hawkins et al. , J. Mol. Biol. , 226:889-896 (1992); Clackson et al. , Nature, 352:624-628 (1991); Gram et al. , Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al. , Bio/Technology, 9:1373-1377 (1991); Hoogenboom et al. , Nucl. Acids Res. , 19:4133-4137 (1991); Barbas et al. , Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); U.S. Patent Application Publication No. 2003/0186374; and PCT Publication No. WO 97/29131.

The recombinant antibody library can be derived from a subject immunized with GFAP or a portion of GFAP. Alternatively, the recombinant antibody library can be derived from a naive subject (i.e., a subject not immunized with GFAP), such as a human antibody library derived from a human subject not immunized with human GFAP. The antibodies of the present invention are selected by screening the recombinant antibody library with a peptide containing human GFAP to select an antibody that recognizes GFAP. Methods for performing such screening and selection are well known in the art, as described in the references listed in the previous paragraph. To select an antibody of the present invention having a specific binding affinity for GFAP (e.g., an antibody that dissociates from human GFAP with a specific K _off rate constant), surface plasmon resonance methods known in the art can be used to select an antibody with a desired K _off rate constant. To select an antibody of the present invention having a specific neutralizing activity against GFAP (e.g., an antibody with a specific IC ₅₀ ), standard methods known in the art for assessing inhibition of GFAP activity can be used.

In one aspect, the invention relates to an isolated antibody or antigen-binding portion thereof that binds to human GFAP. The antibody is preferably a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies can be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that contain polynucleotide sequences encoding said domains. Such phage can be used to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). For example, labeled antigen or antigen bound or captured to a solid surface or bead can be used to antigen-select or identify phage that express an antigen binding domain that binds to the antigen of interest. Phages used in these methods are generally filamentous phage containing fd and M13 binding domains expressed from phages recombinantly fused to phage gene III or gene VIII proteins with Fab antibody domains, Fv antibody domains, or disulfide bond stabilized Fv antibody domains. Examples of phage display methods that can be used to generate antibodies include those described in Brinkmann et al., J. Immunol. Methods, 182:41-50 (1995); Ames et al. , J. Immunol. Methods, 184:177-186 (1995); Kettleborough et al. , Eur. J. Immunol. , 24:952-958 (1994); Persic et al. , Gene, 187:9-18 (1997); Burton et al. ,Advances in Immunology, 57:191-280 (1994); PCT Publication No. WO 92/01047; PCT Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and U.S. Pat. Nos. 5,698,426, 5,2 Examples of the methods disclosed in the above-mentioned patents include those disclosed in Nos. 23,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108.

After phage selection, as described in the above references, antibody encoding regions from the phage can be isolated and used to generate full length antibodies, including human antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, for example, as described in more detail below (see, e.g., PCT Publication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34:26-34 (1995); and Better et al., J. Immunol., 34:26-34 (1995)). Techniques are also available for recombinant production of Fab, Fab', and F(ab') ₂ fragments using methods known in the art, such as those disclosed in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., J. Immunol. 1999, 11:1112-1113 (1994). Examples of techniques that can be used to make single chain Fv and antibodies include those disclosed in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology, 203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. USA, 90:7995-7999 (1993); and Skerra et al., J. Immunol. 1999, 11:1112-1113 (1994). , Science, 240:1038-1041 (1988).

As an alternative to screening recombinant antibody libraries using phage display techniques, other methods known in the art for screening large combinatorial libraries can also be applied to identify antibodies of the invention. One example of an alternative expression system is the expression of recombinant antibody libraries as RNA-protein fusions, as described in PCT Publication No. WO 98/31700 (Szostak and Roberts) and Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297-12302 (1997). In this system, in vitro translation of synthetic mRNAs with the peptidyl acceptor antibiotic puromycin attached to their 3' ends creates covalent fusions of the mRNAs and the peptides or proteins they encode. Thus, specific mRNAs can be enriched from a complex mixture of mRNAs (e.g., a combinatorial library) based on the properties of the encoded peptide or protein (e.g., an antibody or portion thereof) (e.g., binding of the antibody or portion thereof to a dual-specific antigen). Nucleic acid sequences encoding antibodies or portions thereof recovered from screening of such libraries can be expressed by recombinant means (e.g., in mammalian host cells) as described above and further subjected to affinity maturation by screening additional rounds of mRNA-peptide fusions that introduce mutations into the originally selected sequence, as described above, or by other in vitro affinity maturation methods of recombinant antibodies. A preferred example of this method is the PROfusion display technology.

In another approach, antibodies can be made using yeast display methods known in the art, in which antibody domains are tethered to the yeast cell wall using genetic engineering techniques and displayed on the surface of the yeast. Such yeast can be used to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or mouse). Examples of yeast display methods that can be used to make antibodies include those disclosed in U.S. Patent No. 6,699,658 (Wittrup et al.), which is incorporated herein by reference.

d. Production of Recombinant GFAP Antibodies Antibodies can be produced by any of a number of techniques known in the art. For example, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques and expressed from the host cell. Variations of the term "transfection" are intended to encompass a variety of techniques commonly used for introducing foreign DNA into prokaryotic or eukaryotic host cells, including, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although the antibodies of the invention can be expressed in either prokaryotic or eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, with mammalian host cells being most preferred, since eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete a correctly folded, immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) used with the DHFR selection marker, as described, for example, in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982) (including dhfr-CHO cells as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), NS0 myeloma cells, COS cells, and SP2 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into the mammalian host cells, the antibody is produced by culturing the host cells for a time sufficient to allow expression of the antibody in the host cells, or more preferably, secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods.

Host cells can also be used to generate functional antibody fragments, such as Fab fragments or scFv molecules. Of course, variations on the above procedures can be implemented. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of either the light and/or heavy chains of the antibodies of the invention. Recombinant DNA techniques can be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also included in the antibodies of the invention. Furthermore, bifunctional antibodies can be generated in which one heavy and light chain is an antibody of the invention (i.e., binds human GFAP) and the other heavy and light chains are specific for an antigen other than human GFAP, by crosslinking the antibodies of the invention with a second antibody by standard chemical crosslinking methods.

In a preferred recombinant expression system for the antibody or antigen-binding portion thereof of the present invention, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate transfection. Inside the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLP promoter regulatory element, driving high levels of transcription of said genes. Because the recombinant expression vector also incorporates a DHFR gene, methotrexate selection/amplification can be used to select CHO cells transfected with the vector. The selected transformed host cells are cultured to express the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to generate the recombinant expression vector, transfect the host cells, select the transformed cells, culture the host cells, and recover the antibody from the culture medium. The present invention further provides a method for synthesizing a recombinant antibody of the present invention by culturing the host cells of the present invention in an appropriate culture medium until a recombinant antibody of the present invention is synthesized. The method may further include isolating the recombinant antibody from the culture medium.

(1) Humanized Antibodies A humanized antibody can be an antibody, or a variant, derivative, analog or portion thereof, that immunospecifically binds to an antigen of interest and contains a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody can be derived from a non-human species antibody that binds a desired antigen and has one or more complementarity determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

The term "substantially" as used herein with respect to CDRs refers to a CDR having an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab') ₂ , FabC, Fv), in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are framework regions of a human immunoglobulin consensus sequence. According to one aspect, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. In some embodiments, the humanized antibody comprises both a light chain and at least the variable domain of a heavy chain. The antibody further comprises the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only comprises a humanized light chain. In some embodiments, a humanized antibody only comprises a humanized heavy chain. In certain embodiments, a humanized antibody only comprises humanized variable domains of the light and/or heavy chain.

Humanized antibodies can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. Humanized antibodies can contain sequences from more than one class or isotype, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion, and/or deletion of at least one amino acid residue such that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in one embodiment, such mutations will not be extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of cognate immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either may be included in the consensus sequence.

Immune responses to rodent anti-human antibodies limit the duration and effectiveness of therapeutic applications of these moieties in human recipients, and humanized antibodies can be designed to minimize such undesirable immune responses. Humanized antibodies can have one or more amino acid residues introduced from a non-human source. These non-human residues are often referred to as "import" residues and are typically taken from the variable domain. Humanization can be performed by substituting hypervariable region sequences for corresponding sequences in a human antibody. Such "humanized" antibodies are thus chimeric antibodies in which substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. See, for example, U.S. Pat. No. 4,816,567, the disclosure of which is incorporated herein by reference. Humanized antibodies can be human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies. Humanization or engineering of the antibodies of the present invention can be carried out using any known method, including, but not limited to, those described in U.S. Patent Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, and 4,816,567.

Humanized antibodies can maintain high affinity for GFAP and other favorable biological properties. Humanized antibodies can be generated by analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are widely available. Computer programs are available that draw and display predicted three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the predicted role of the residues in the function of the candidate immunoglobulin sequence, i.e., residues that influence the ability of the candidate immunoglobulin to bind its antigen. FR residues can thus be selected and combined from the recipient and import sequences to obtain desired antibody characteristics, such as increased affinity for GFAP. In general, it appears that the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

As an alternative to humanization, human antibodies (also referred to herein as "fully human antibodies") can be produced. For example, human antibodies can be isolated from libraries by PROfusion and/or yeast-related technologies. Transgenic animals (e.g., mice capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production after immunization) can also be produced. For example, the homozygous deletion of the antibody heavy-chain joining region ( _JH ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germ-line immunoglobulin gene sequences into such germ-line mutant mice will result in the production of human antibodies after antigen challenge. Humanized or fully human antibodies can be made according to the methods described in U.S. Pat. Nos. 5,770,429, 5,833,985, 5,837,243, 5,922,845, 6,017,517, 6,096,311, 6,111,166, 6,270,765, 6,303,755, 6,365,116, 6,410,690, 6,682,928, and 6,984,720, the disclosures of each of which are incorporated herein by reference.

e. Anti-GFAP Antibodies Anti-GFAP antibodies can be made using the techniques described above and other conventional techniques known in the art. In certain embodiments, anti-GFAP antibodies are available from Dako (catalog number: M0761), ThermoFisher Scientific (catalog numbers: MA5-12023, A-21282, 13-0300, MA1-19170, MA1-19395, MA5-15086, MA5-16367, MA1-35377, MA1-06701, or MA1-20035), AbCam (catalog numbers: ab10062, ab4648, ab68428, ab33922, ab207165, ab190288, ab115898, or ab21837), EMD The antibody can be an unconjugated GFAP antibody, such as those available from Millipore (catalog numbers: FCMAB257P, MAB360, MAB3402, 04-1031, 04-1062, MAB5628), Santa Cruz (catalog numbers: sc-166481, sc-166458, sc-58766, sc-56395, sc-51908, sc-135921, sc-71143, sc-65343, or sc-33673), Sigma-Aldrich (catalog numbers: G3893 or G6171), or Sino Biological Inc. (catalog number: 100140-R012-50). The anti-GFAP antibody may be conjugated to a fluorophore, such as the conjugated GFAP antibodies available from ThermoFisher Scientific (Cat. Nos. A-21295 or A-21294), EMD Millipore (Cat. Nos. MAB3402X, MAB3402B, MAB3402B, or MAB3402C3) or AbCam (Cat. Nos. ab49874 or ab194325).

8. Method Variations The present application discloses a method for determining the presence or amount of an analyte of interest (UCH-L1 and/or GFAP) present in a sample, which may be carried out as described herein. The method may be modified to accommodate other methods of analysis of the analyte. Examples of well-known variations include, but are not limited to, sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, including enzyme detection methods (enzyme immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA)), monoclonal-polyclonal sandwich immunoassays), competitive inhibition immunoassays (e.g., forward and reverse), immunoassays such as multiple enzyme immunoassay technology (EMIT), competitive binding assays, bioluminescence resonance energy transfer (BRET), one-step antibody detection assays, homogeneous assays, heterogeneous assays, capture on the fly assays, and the like.

a. Immunoassays Analytes of interest and/or peptides or fragments thereof (e.g., UCH-L1 and/or GFAP, and/or peptides or fragments thereof, i.e., UCH-L1 and/or GFAP fragments) can be analyzed using UCH-L1 and/or GFAP antibodies in an immunoassay. The antibodies can be used to measure the presence or amount of the analyte (e.g., UCH-L1 and/or GFAP) by detecting specific binding to the analyte (e.g., UCH-L1 and/or GFAP). For example, an antibody or antibody fragment thereof can specifically bind to the analyte (e.g., UCH-L1 and/or GFAP). If desired, one or more of the above antibodies can be used in combination with one or more commercially available monoclonal/polyclonal antibodies. Such antibodies are available from R&D Systems, Inc. (Minneapolis, Minn.) and Enzo Life Sciences International, Inc. (Plymouth Meeting, Pa.).

The presence or amount of an analyte (e.g., UCH-L1 and/or GFAP) present in a biological sample can be readily measured using immunoassays such as sandwich immunoassays (e.g., monoclonal-monoclonal sandwich immunoassays, monoclonal-polyclonal sandwich immunoassays, including radioisotope detection methods (radioimmunoassays (RIA)) and enzyme detection methods (enzyme immunoassays (EIA) or enzyme immunoassays (ELISA) (e.g., Quantikine ELISA assay, R&D Systems, Minneapolis, Minn.)). One example of a point-of-care device that can be used is i-STAT® (Abbott Laboratories, Abbott Park, Ill.). Other methods that can be used include chemiluminescent microparticle immunoassays, one example of which is utilizing an ARCHITECT® automated analyzer (Abbott Laboratories, Abbott Park, Ill.), mass spectrometry using, for example, anti-analyte (e.g., anti-UCH-L1 and/or anti-GFAP) antibodies (monoclonal, polyclonal, chimeric, humanized, human, etc.) or antibody fragments thereof against the analyte (e.g., UCH-L1 and/or GFAP), and immunohistochemistry (e.g., using sections from tissue biopsies). Other detection methods include those described in, for example, U.S. Patent Nos. 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615, 5,885,527, 5,851,776, 5,824,799, 5,679,526, 5,525,524, and 5,480,792, the disclosures of each of which are incorporated herein in their entirety. Specific immunological binding between the antibody and the analyte (e.g., UCH-L1 and/or GFAP) can be detected by direct labels, such as fluorescent or luminescent tags, metals and radionuclides, or indirect labels, such as alkaline phosphatase or horseradish peroxidase, attached to the antibody.

The use of immobilized antibodies or antibody fragments thereof may be incorporated into immunoassays. Antibodies can be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as a microtiter well), or a small piece of solid substrate material. An assay strip can be made by coating an antibody or multiple antibodies in an array onto a solid support. This strip can then be dipped into the test sample and briefly processed through washing and detection steps to produce a measurable signal, such as a colored spot.

Homogeneous formats may also be used. For example, after obtaining a test sample from a subject, a mixture is prepared. The mixture includes the test sample to be assessed for an analyte (e.g., UCH-L1 and/or GFAP), a first specific binding partner, and a second specific binding partner. The order in which the test sample, the first specific binding partner, and the second specific binding partner are added to form the mixture is not important. The test sample is contacted with the first specific binding partner and the second specific binding partner simultaneously. In certain embodiments, the first specific binding partner and UCH-L1 and/or GFAP contained in the test sample can form a complex of the first specific binding partner, the analyte (e.g., UCH-L1 and/or GFAP) and an antigen, and the second specific binding partner can form a complex of the first specific binding partner, the analyte of interest (e.g., UCH-L1 and/or GFAP) and the second specific binding partner. In certain embodiments, the second specific binding partner and UCH-L1 and/or GFAP contained in the test sample can form an antigen complex between the second specific binding partner and the analyte (e.g., UCH-L1), and the first specific binding partner can form an antigen complex between the first specific binding partner and the analyte of interest (e.g., UCH-L1 and/or GFAP) and the second specific binding partner. The first specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence that includes at least three consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence that includes at least three consecutive amino acids of SEQ ID NO:2). The second specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence that includes at least three consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence that includes at least three consecutive amino acids of SEQ ID NO:2). Additionally, the second specific binding partner is labeled with or contains a detectable label as described above.

Heterogeneous formats may also be used. For example, after obtaining a test sample from a subject, a first mixture is prepared. The mixture includes the test sample to be evaluated for an analyte (e.g., UCH-L1 and/or GFAP) and a first specific binding partner, and the first specific binding partner and the UCH-L1 and/or GFAP contained in the test sample form a complex of the first specific binding partner and the analyte (e.g., UCH-L1 and/or GFAP) antigen. The first specific binding partner may be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence including at least 3 consecutive amino acids of SEQ ID NO: 1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence including at least 3 consecutive amino acids of SEQ ID NO: 2). The order in which the test sample and the first specific binding partner are added to form the mixture is not important.

The first specific binding partner may be immobilized on a solid phase. The solid phase used in the immunoassay (for the first specific binding partner and optionally the second specific binding partner) can be any solid phase known in the art, including but not limited to magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, films, filter paper, disks, and chips. In embodiments where the solid phase is a bead, the bead can be a magnetic bead or magnetic particle. The magnetic bead/particle can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or magnetic fluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, _CrO2 , MnAs, MnBi, EuO, and NiO/Fe. Examples of _{ferrimagnetic} materials include _NiFe2O4 , _CoFe2O4 , _Fe3O4 (or _{FeO.Fe2O3 )} . The beads may have a magnetic solid core portion around which one or _more non-magnetic layers are disposed. Alternatively, the magnetic portion may be a layer around the non-magnetic core. The solid support with the first specific binding member immobilized _thereon may be stored dry or in liquid. A magnetic field may be applied to the magnetic beads before or after the sample is contacted with the magnetic beads with the first specific binding member immobilized thereon.

After a mixture containing a complex of the first specific binding partner and the analyte (e.g., UCH-L1 or GFAP) antigen is formed, any technique known in the art is used to remove unbound analyte (e.g., UCH-L1 and/or GFAP) from the complex. For example, unbound analyte (e.g., UCH-L1 and/or GFAP) can be removed by washing. However, it is desirable that the first specific binding partner is present in excess of the analyte (e.g., UCH-L1 and/or GFAP) present in the test sample, so that all of the analyte (e.g., UCH-L1 and/or GFAP) present in the test sample binds to the first specific binding partner.

After removing unbound analyte (e.g., UCH-L1 and/or GFAP), a second specific binding partner is added to the mixture to form a complex of the first specific binding partner, the analyte of interest (e.g., UCH-L1 and/or GFAP), and the second specific binding partner. The second specific binding partner can be an anti-analyte antibody (e.g., an anti-UCH-L1 antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO:1 or an anti-GFAP antibody that binds to an epitope having an amino acid sequence that includes at least 3 consecutive amino acids of SEQ ID NO:2). Additionally, the second specific binding partner is labeled with or includes a detectable label as described above.

The use of immobilized antibodies or antibody fragments thereof may be incorporated into immunoassays. Antibodies can be immobilized on a variety of supports, such as magnetic or chromatographic matrix particles (e.g., magnetic beads), latex particles or surface-modified latex particles, polymers or polymer films, plastics or plastic films, planar substrates, the surface of an assay plate (e.g., microtiter wells), or strips of solid substrate material. An assay strip can be made by coating an antibody or multiple antibodies in an array onto a solid support. This strip can then be dipped into the test sample and briefly processed through washing and detection steps to produce a measurable signal, such as a colored spot.

In certain embodiments, it is contemplated that other antibodies can be selected that can similarly help maintain the dynamic range and low-end sensitivity of the immunoassay. For example, it may be useful to select at least one first antibody (such as a capture antibody or first specific binding partner) that binds to an epitope near the N-terminus of 38 kDa BDP and at least one second antibody (such as a detection antibody or second specific binding partner) that binds to an epitope near the center of 38 kDa BDP (e.g., near the center of 38 kDa BDP) and does not overlap with the first antibody. Other variations are contemplated and can be readily tested by one of ordinary skill in the art, such as by screening antibody pairs using calibrator concentrations to identify antibodies that bind to different epitopes, after testing for binding to short peptides. Additionally, methods of selecting antibodies with different affinities for GFAP can also help maintain or increase the dynamic range of the assay. GFAP antibodies have been described in the literature and are commercially available.

(1) Sandwich Immunoassay Sandwich immunoassays measure the amount of antigen between two layers of antibodies (i.e., at least one capture antibody) and detection antibodies (i.e., at least one detection antibody). The capture antibody and detection antibody bind to different epitopes on the antigen (e.g., an analyte of interest such as UCH-L1 and/or GFAP). It is desirable that the binding of the capture antibody to the epitope does not interfere with the binding of the detection antibody to the epitope. In sandwich immunoassays, either monoclonal or polyclonal antibodies may be used as the capture antibody and detection antibody.

Typically, at least two antibodies are utilized to separate and quantify an analyte (e.g., UCH-L1 and/or GFAP) in a test sample. More specifically, the at least two antibodies bind to a predefined epitope of the analyte (e.g., UCH-L1 and/or GFAP) to form an immune complex called a "sandwich." One or more antibodies can be used to capture the analyte (e.g., UCH-L1 and/or GFAP) in the test sample (these antibodies are often referred to as a "capture" antibody or antibodies), and one or more antibodies are used to bind a detectable (i.e., quantifiable) label to the sandwich (these antibodies are often referred to as a "detection" antibody or antibodies). In a sandwich assay, it is desirable that the binding of the antibody to its epitope is not weakened by the binding of other antibodies in the assay to their respective episomes. The antibodies are selected so that one or more first antibodies contacted with a test sample suspected of containing an analyte (e.g., UCH-L1 and/or GFAP) do not bind to all or part of the epitope recognized by the second or subsequent antibodies, and do not interfere with the binding of one or more second detection antibodies to the analyte (e.g., UCH-L1 and/or GFAP).

In the immunoassay, the antibody of the present application can be used as a first antibody. The antibody immunospecifically binds to an epitope on the analyte (e.g., UCH-L1 and/or GFAP). In addition to the antibody of the present invention, the immunoassay can include a second antibody that immunospecifically binds to an epitope not recognized or bound by the first antibody.

A test sample suspected of containing an analyte (e.g., UCH-L1 and/or GFAP) can be contacted simultaneously or sequentially with at least one first capture antibody (or multiple capture antibodies) and at least one second detection antibody. In a sandwich assay format, a test sample suspected of containing an analyte (e.g., UCH-L1 and/or GFAP) is first contacted with at least one first capture antibody that specifically binds to a particular epitope under conditions that allow the formation of a complex between the first antibody and the analyte (e.g., UCH-L1 and/or GFAP) antigen. When more than one capture antibody is used, a complex between the first multiple capture antibodies and the UCH-L1 and/or GFAP antigen is formed. In a sandwich assay, the antibody of the present application, preferably the at least one capture antibody, is used in a molar excess over the maximum amount of analyte (e.g., UCH-L1 and/or GFAP) expected in the test sample. For example, about 5 μg/mL to about 1 mg/mL of antibody per ml of microparticle coating buffer can be used.

i. Anti-UCH-L1 Capture Antibody Optionally, prior to contacting the test sample with the at least one first capture antibody, the at least one first capture antibody can be immobilized on a solid support to facilitate separation of the complex of the first antibody and the analyte (e.g., UCH-L1 and/or GFAP) from the test sample. Any solid support known in the art can be used, including, but not limited to, solid supports made of polymeric materials in the form of wells, tubes, or beads (e.g., microparticles). The antibody (or antibodies) can be immobilized on the solid support by adsorption, by covalent attachment using chemical coupling agents, or by other means known in the art, provided that the antibody is not prevented from binding to the analyte (e.g., UCH-L1 and/or GFAP). Additionally, the solid support can be derivatized, if desired, to be reactive to various functional groups on the antibodies. Such derivatization requires the use of certain coupling agents, including, but not limited to, maleic anhydride, N-hydroxysuccinimide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Then, the test sample suspected of containing the analyte (e.g., UCH-L1 and/or GFAP) is incubated to allow for the formation of a complex between the first capture antibody (or antibodies) and the analyte (e.g., UCH-L1 and/or GFAP). The incubation can be carried out at a pH of about 4.5 to about 10.0 and at a temperature of about 2°C to about 45°C for at least about 1 minute to about 18 hours, about 2-6 minutes, about 7-12 minutes, about 5-15 minutes, or about 3-4 minutes.

ii. Detection Antibodies Following formation of a complex between the first/plurality of capture antibodies and the analyte (e.g., UCH-L1 and/or GFAP), the complex is then contacted with at least one second detection antibody (under conditions that allow for the formation of a complex between the first/plurality of antibodies, the analyte (e.g., UCH-L1 and/or GFAP) antigen, and the second antibody). In certain embodiments, the test sample is contacted with the detection antibody simultaneously with the capture antibody. When the first antibody/analyte (e.g., UCH-L1 and/or GFAP) complex is contacted with more than one detection antibody, complexes between the first/plurality of capture antibodies, the analyte (e.g., UCH-L1 and/or GFAP) and multiple detection antibodies are formed. As with the first antibody, at least a second (or subsequent) antibody is contacted with the first antibody-analyte (e.g. UCH-L1 and/or GFAP) complex under similar conditions as described above and for the incubation time required for the formation of a complex of the first antibody/several antibodies-analyte (e.g. UCH-L1 and/or GFAP) and the second/several antibodies. Preferably, the at least one second antibody comprises a detectable label. The detectable label may be attached to the at least one second antibody before, simultaneously with, or after the formation of the complex of the first antibody/several antibodies-analyte (e.g. UCH-L1 and/or GFAP) and the second/several antibodies. Any detectable label known in the art may be used.

Chemiluminescence assays can be performed according to the methods described in Adamczyk et al., Anal. Chim. Acta 579(1):61-67 (2006). While any suitable assay format can be used, a microplate chemiluminometer (Mithras LB-940, Berthold Technologies U.S.A., LLC, Oak Ridge, Tenn.) can be used to rapidly assay multiple small samples. The chemiluminometer uses 96-well black polystyrene microplates (Costar #3792) and can be equipped with multiple reagent injectors. Each sample is added to a separate well, and other reagents can be added simultaneously/sequentially depending on the type of assay being utilized. It is desirable to prevent pseudobase formation in neutral or basic solutions utilizing acridinium aryl esters, such as by acidification. The chemiluminescence response is then recorded for each well. In this regard, the time to record the chemiluminescent response will also vary depending on the reagent utilized and the delay between addition of the particular acridinium.

The order in which the test sample and the specific binding partner are added to form the chemiluminescent assay mixture is not important. When the first specific binding partner is detectably labeled with an acridinium compound, a detectably labeled complex of the first specific binding partner and the antigen (e.g., UCH-L1 and/or GFAP) is formed. Alternatively, when a second specific binding partner is used and the second specific binding partner is detectably labeled with an acridinium compound, a detectably labeled complex of the first specific binding partner and the analyte (e.g., UCH-L1 and/or GFAP) is formed. Unbound specific binding partners, whether labeled or not, can be removed from the mixture using any technique known in the art, such as washing.

Hydrogen peroxide can be generated in situ in the mixture or provided or delivered to the mixture before, simultaneously with, or after the addition of the acridinium compound. Hydrogen peroxide can be generated in situ in a number of ways, as will be apparent to those skilled in the art.

Alternatively, one can simply add a source of hydrogen peroxide to the mixture. For example, the source of hydrogen peroxide can be one or more buffers or other solutions known to contain hydrogen peroxide. In this regard, one can simply add a hydrogen peroxide solution.

After simultaneously or subsequently adding at least one basic solution to the sample, a detectable signal (i.e., a chemiluminescent signal) is generated that indicates the presence of the analyte (e.g., UCH-L1 and/or GFAP). The basic solution contains at least one base and has a pH of 10 or higher, preferably 12 or higher. Examples of basic solutions include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium hydroxide, calcium carbonate, and calcium bicarbonate. The amount of basic solution added to the sample depends on the concentration of the basic solution. Based on the concentration of the basic solution used, one of skill in the art can easily determine the amount of basic solution to add to the sample. Labels other than chemiluminescent labels can also be used. For example, enzyme labels (including but not limited to alkaline phosphatase) can be used.

The chemiluminescent or other signal generated can be detected using conventional techniques known to those of skill in the art. Based on the intensity of the signal generated, the amount of analyte of interest (e.g., UCH-L1 and/or GFAP) in the sample can be quantified. Specifically, the amount of analyte (e.g., UCH-L1 and/or GFAP) in the sample is proportional to the intensity of the signal generated. The amount of analyte (e.g., UCH-L1 and/or GFAP) present can be quantified by comparing the amount of light generated to a standard curve of the analyte (e.g., UCH-L1 and/or GFAP) or by comparing to a reference standard. The standard curve can be generated using serial dilutions or solutions of known concentrations of the analyte (e.g., UCH-L1 and/or GFAP) by mass spectrometry, gravimetry, or other techniques known in the art.

(2) Forward competitive inhibition assay In the forward competitive format, a labeled aliquot of known concentration of an analyte of interest (e.g., an analyte (e.g., UCH-L1 and/or GFAP) attached with a fluorescent label, a tag attached to a cleavable linker, etc.) is used to compete with the analyte of interest (e.g., UCH-L1 and/or GFAP) in a test sample for binding to an antibody of the analyte of interest (e.g., a UCH-L1 and/or GFAP antibody).

In a forward competitive assay, an immobilized specific binding partner (e.g., an antibody) can be contacted sequentially or simultaneously with the test sample and a labeled analyte of interest, analyte fragment of interest, or analyte variant of interest thereof. The analyte peptide of interest, analyte fragment of interest, or analyte variant of interest can be labeled with any detectable label, including a detectable label consisting of a tag coupled to a cleavable linker. In this assay, the antibody can be immobilized on a solid support. Alternatively, the antibody can be coupled to an antibody, such as an anti-species antibody, that is immobilized on a solid support, such as a microparticle or planar substrate.

The labeled analyte of interest, the test sample and the antibody are incubated under similar conditions as described above in connection with the sandwich assay format. In this way, two different types of antibody-analyte of interest complexes can be generated. Specifically, one of the generated antibody-analyte of interest complexes contains a detectable label (e.g., a fluorescent label, etc.) and the other antibody-analyte of interest complex does not contain a detectable label. Prior to quantifying the detectable label, the antibody-analyte of interest complex can, but does not necessarily, be separated from the remainder of the test sample. Whether or not the antibody-analyte of interest complex is separated from the remainder of the test sample, the amount of detectable label in the antibody-analyte of interest complex is then quantified. The concentration of the analyte of interest (e.g., membrane-bound analyte of interest, soluble analyte of interest, fragment of soluble analyte of interest, mutant of analyte of interest (membrane-bound or soluble analyte of interest), or any combination thereof) in the test sample can then be measured, for example as described above.

(3) Reverse competitive inhibition assay In a reverse competitive assay, a solid-phase analyte of interest (e.g., UCH-L1 and/or GFAP) can be contacted sequentially or simultaneously with a test sample and at least one type of labeled antibody.

The analyte can be immobilized on a solid phase, such as those described above in connection with the sandwich assay format.

The immobilized analyte of interest, the test sample, and at least one labeled antibody are incubated under similar conditions as described above in connection with the sandwich assay format. In this way, two different types of analyte of interest-antibody complexes are generated. Specifically, one of the generated analyte of interest-antibody complexes is immobilized and contains a detectable label (e.g., a fluorescent label, etc.), and the other analyte of interest-antibody complex is not immobilized and contains a detectable label. The non-immobilized analyte of interest-antibody complex and test sample residues are removed from the immobilized analyte of interest-antibody complex by techniques known in the art, such as washing. Once the non-immobilized analyte of interest-antibody complex has been removed, the amount of detectable label in the immobilized analyte of interest-antibody complex is then quantified after tag cleavage. The amount of detectable label can then be compared as described above to determine the concentration of the analyte of interest in the test sample.

(4) One-Step Immunoassays or "Capture-on-the-Fly" Assays In capture-on-the-fly immunoassays, the solid support is pre-coated with a solid-phase immobilizing agent. The capture agent, analyte (e.g., UCH-L1 and/or GFAP) and detection agent are added together to the solid support, followed by a wash step prior to detection. The capture agent is capable of binding to the analyte (e.g., UCH-L1 and/or GFAP) and includes a ligand for the immobilizing agent. The capture agent and detection agent can be antibodies or any other capture or detectable moiety as described herein or known in the art. The ligand can include a peptide tag and the immobilizing agent can include an anti-peptide tag antibody. Alternatively, the ligand and immobilizing agent can be any pair of substances capable of binding to each other (e.g., specific binding pairs and other pairs known in the art) to be utilized in a capture-on-the-fly assay. More than one analyte can be measured. In certain embodiments, the solid support is coated with an antigen and the analyte to be analyzed is an antibody.

In certain other embodiments, a one-step immunoassay or "capture-on-the-fly" assay uses a solid support (such as a microparticle) pre-coated with an immobilizing agent (such as biotin, streptavidin, etc.) and at least a first specific binding member and a second specific binding member (which function as a capture agent and a detection reagent, respectively). The first specific binding member includes a ligand of the immobilizing agent (e.g., if the immobilizing agent on the solid support is streptavidin, the ligand on the first specific binding member can be biotin) and further binds to the analyte of interest (e.g., UCH-L1 and/or GFAP). The second specific binding member includes a detectable label and binds to the analyte of interest (e.g., UCH-L1 and/or GFAP). The solid support and the first and second specific binding members can be added (sequentially or simultaneously) to a test sample. The ligand on the first specific binding member binds to the immobilizing agent on the solid phase carrier, forming a complex between the solid phase carrier and the first specific binding member. If an analyte of interest is present in the sample, it binds to the complex between the solid phase carrier and the first specific binding member, forming a complex between the solid phase carrier and the first specific binding member and the analyte. The second specific binding member binds to the complex between the solid phase carrier and the first specific binding member and the analyte and the detectable label is detected. An optional wash step may be utilized prior to detection. In certain embodiments, more than one analyte may be measured in the one-step assay. In certain other embodiments, more than one specific binding member may be utilized. In certain other embodiments, multiple detectable labels may be added. In certain other embodiments, multiple analytes of interest may be detected or the amount, value or concentration may be measured, determined or assessed.

The capture-on-the-fly assays described herein can be used in a variety of formats known in the art. For example, the format can be a sandwich assay as described above, or it can be a competitive assay, can utilize a single specific binding member, or can use other variations known in the art.

9. Other Factors The above diagnostic, prognostic and/or assessment methods further include the use of other factors for diagnosis, prognosis and assessment. In certain embodiments, the Glasgow Coma Scale can be used to diagnose traumatic brain injury. Other tests, scales or indices can be used alone or in combination with the Glasgow Coma Scale. One example is the Ranchos Los Amigos Scale. The Ranchos Los Amigos Scale measures levels of consciousness, cognition, behavior and response to the environment. The Ranchos Los Amigos Scale includes the following levels: Level I: no response; Level II: generalized response; Level III: localized response; Level IV: confusion-agitation; Level V: confusion-inappropriate; Level VI: confusion-appropriate; Level VII: automatic-appropriate; and Level VIII: intentional-appropriate. Another example is the Rivermead Post-Concussion Symptoms Questionnaire, a self-report scale for measuring the severity of post-concussion symptoms following TBI. Patients are asked to rate how severe each of 16 symptoms (e.g., headache, dizziness, nausea, vomiting) has been during the past 24 hours. In each case, the symptom is compared to its severity before the injury occurred (premorbidity). These symptoms are reported by severity on a scale of 0 to 4: no symptoms, no impairment, mild impairment, moderate impairment, and severe impairment.

10. Samples In certain embodiments, a sample is obtained after a subject, such as a human subject, experiences a head injury resulting from bodily shaking, blunt impact from an external mechanical or other force resulting in a closed or open head injury, one or more falls, an explosion or blast, or other type of blunt trauma. In certain embodiments, a sample is obtained after a subject, such as a human subject, inhales or is exposed to a fire, a chemical, a toxin, or a combination of fire, chemicals, and toxins. Examples of such chemicals and/or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (e.g., carbon monoxide, hydrogen sulfide, and cyanides), organometallics (e.g., methylmercury, tetraethyl lead, and organotins), and/or one or more drugs of abuse. In certain embodiments, the sample is obtained from a subject, such as a human subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.

In yet another embodiment, the methods described herein use a sample that can also be used to determine whether a subject has or is at risk for developing TBI (e.g., mild TBI, moderate TBI, severe TBI, or moderate-severe TBI) by measuring the levels of UCH-L1 and/or GFAP in the subject using anti-UCH-L1 and/or anti-GFAP antibodies or antibody fragments thereof described below. Thus, in certain embodiments, the present disclosure also provides methods for determining whether a subject having or at risk for traumatic brain injury as described herein and known in the art is a candidate for a therapy or treatment. In general, the subject is a subject who, as described herein, at least (i) has suffered a head injury; (ii) has inhaled and/or been exposed to one or more chemicals and/or toxins; (iii) suffers from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof; or (iv) any combination of (i)-(iii), or has actually been diagnosed as having or at risk for TBI (e.g., a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection (e.g., SARS-CoV-2), a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof), and/or exhibits unfavorable (i.e., clinically undesirable) concentrations or amounts of UCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragments.

a. Test Sample or Biological Sample As used herein, "sample", "test sample", "biological sample" refers to a liquid sample that contains or is suspected of containing UCH-L1 and/or GFAP. The sample may be from any suitable source. Optionally, the sample may comprise a liquid, a flowable granular solid, or a suspension of solid particles. Optionally, the sample may be processed prior to the analysis described herein. For example, the sample may be separated or purified from its source prior to analysis, although in certain embodiments, unprocessed samples containing UCH-L1 and/or GFAP may be assayed directly. In certain instances, the source of UCH-L1 and/or GFAP is human biological material (e.g., bodily fluids, blood such as whole blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph, amniotic fluid, interstitial fluid, lung lavage, cerebrospinal fluid, oropharyngeal specimen, nasopharyngeal specimen, feces, tissue, organ, etc.). Tissues include, but are not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, cardiac muscle tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample may be a liquid sample or an extract of a solid sample. In some cases, the source of the sample may be an organ or tissue, such as a biopsy sample, and may be solubilized by tissue digestion/cell lysis.

A wide range of liquid sample volumes can be analyzed. In a few exemplary embodiments, the sample volume can be about 0.5 nL, about 1 nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL, about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, etc. In some cases, the volume of the bodily fluid sample can be about 0.01 μL to about 10 mL, about 0.01 μL to about 1 mL, about 0.01 μL to about 100 μL, or about 0.1 μL to about 10 μL.

Optionally, the liquid sample may be diluted prior to use in the assay. For example, in embodiments where the source of UCH-L1 and/or GFAP is a human body fluid (e.g., blood, serum), the body fluid may be diluted with an appropriate solvent (e.g., a buffer such as PBS buffer). The liquid sample may be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more prior to use. In other cases, the liquid sample is not diluted prior to use in the assay.

Optionally, the sample may be subjected to pre-analysis treatment. Pre-analysis treatment may provide additional functionality, such as non-specific protein removal and/or mixing functions that are effective and inexpensive to implement. Common methods of pre-analysis treatment include the use of electrokinetic trapping, AC electrokinetic action, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other pre-concentration techniques known in the art. Optionally, the liquid sample may be concentrated before use in the assay. For example, in embodiments where the source of UCH-L1 and/or GFAP is a human body fluid (e.g., blood, serum), the body fluid may be concentrated by precipitation, evaporation, filtration, centrifugation, or combinations thereof. The liquid sample may be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more, prior to use.

b. Controls It may be desirable to include a control sample. The control sample may be analyzed simultaneously with the sample from the subject as described above. Results from the subject sample may be compared to results from the control sample. A standard curve may be prepared against which the assay results of the samples may be compared. Such a standard curve would show the marker value (i.e., fluorescent signal intensity, if a fluorescent label is used) as a function of assay units. Using samples from multiple donors, standard curves may be prepared for reference values of UCH-L1 and/or GFAP in normal healthy tissue and "risk" values of UCH-L1 and/or GFAP in tissue from donors likely to have one or more of the above characteristics.

Thus, in view of the above, there is provided a method for determining the presence, amount, or concentration of UCH-L1 and/or GFAP in a test sample, the method comprising, for example, assaying the test sample for UCH-L1 and/or GFAP by immunoassay using at least one capture antibody that binds to an epitope on UCH-L1 and/or GFAP and at least one detection antibody that binds to an epitope on UCH-L1 and/or GFAP that is distinct from the epitope of the capture antibody and optionally includes a detectable label, and comparing a signal generated by the detectable label as a direct or indirect indication of the presence, amount, or concentration of UCH-L1 and/or GFAP in the test sample to a signal generated in a calibrator as a direct or indirect indication of the presence, amount, or concentration of UCH-L1 and/or GFAP. The calibrators are optionally, and preferably are, part of a calibrator series in which the concentration of UCH-L1 and/or GFAP in each of the calibrators differs from other calibrators in the series.

11. Kits The present application provides kits that can be used to assay or assess a test sample for UCH-L1 and/or GFAP or UCH-L1 and/or GFAP fragments. The kits include at least one component for assaying the test sample for UCH-L1 and/or GFAP and instructions for assaying the test sample for UCH-L1 and/or GFAP. For example, the kits can include instructions for assaying the test sample for UCH-L1 and/or GFAP by immunoassay (e.g., chemiluminescent microparticle immunoassay). The instructions included in the kits can be affixed to the packaging material or can be included as a package insert. The instructions are typically written or printed, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include an address of an internet site providing the instructions.

The at least one component may include at least one composition comprising one or more isolated antibodies or antibody fragments thereof that specifically bind to UCH-L1 and/or GFAP. The antibodies may be UCH-L1 and/or GFAP capture antibodies and/or UCH-L1 and/or GFAP detection antibodies.

Alternatively or additionally, the kit may include a calibrator or control (e.g., purified and optionally lyophilized UCH-L1 and/or GFAP), and/or at least one container for performing the assay (e.g., a tube, microtiter plate or strip that may be pre-coated with anti-UCH-L1 and/or GFAP monoclonal antibody), and/or a buffer, such as an assay buffer or a wash buffer, either of which may be provided as a concentrate solution, a substrate solution for a detectable label (e.g., an enzyme label), or a stop solution. The kit preferably includes all components (i.e., reagents, standards, buffers, diluents, etc.) required to perform the assay. The instructions may further include instructions for generating a standard curve.

The kit may further include a reference standard for quantifying UCH-L1 and/or GFAP. The reference standard may be used to generate a standard curve for interpolation and/or extrapolation of UCH-L1 and/or GFAP concentrations. The reference standards may be high UCH-L1 and/or GFAP concentration values (e.g., about 100,000 pg/mL, about 125,000 pg/mL, about 150,000 pg/mL, about 175,000 pg/mL, about 200,000 pg/mL, about 225,000 pg/mL, about 250,000 pg/mL, about 275,000 pg/mL, or about 300,000 pg/mL), intermediate UCH-L1 and/or GFAP concentration values (e.g., about 25,000 pg/mL, about 40,000 pg/mL, about 45,000 pg/mL, about 50,000 pg/mL, about 55,000 pg/mL, about 60,000 pg/mL, about 75,000 pg/mL, or about 80,000 pg/mL), mL or about 100,000 pg/mL), and/or low UCH-L1 and/or GFAP concentration values (e.g., about 1 pg/mL, about 5 pg/mL, about 10 pg/mL, about 12.5 pg/mL, about 15 pg/mL, about 20 pg/mL, about 25 pg/mL, about 30 pg/mL, about 35 pg/mL, about 40 pg/mL, about 45 pg/mL, about 50 pg/mL, about 55 pg/mL, about 60 pg/mL, about 65 pg/mL, about 70 pg/mL, about 75 pg/mL, about 80 pg/mL, about 85 pg/mL, about 90 pg/mL, about 95 pg/mL, or about 100 pg/mL).

Any antibody, such as a recombinant antibody specific for UCH-L1 and/or GFAP, provided in the kit can be conjugated with a detectable label, such as a fluorophore, a radioactive moiety, an enzyme, a biotin/avidin label, a chromophore, a chemiluminescent label, or the kit can include reagents for labeling or detecting the antibody (e.g., a detection antibody) and/or reagents for labeling or detecting the analyte (e.g., UCH-L1 and/or GFAP). The antibodies, calibrators, and/or controls can be provided in separate containers or pre-distributed in a suitable assay format (e.g., a microtiter plate).

Optionally, the kit includes quality control components (e.g., sensitivity panels, calibrators, and positive controls). Preparation of quality control reagents is well known in the art and is described on various immunodiagnostic product insert sheets. Sensitivity panel members are optionally used to establish assay performance characteristics and are further optionally useful indicators of immunoassay kit reagent integrity and assay standardization.

The kit may further optionally include other reagents (e.g., buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc.) necessary to perform the diagnostic assay or to facilitate quality control evaluation. Other components, such as buffers and solutions for isolating and/or processing the test sample (e.g., pretreatment reagents), may also be included in the kit. The kit may further include one or more other controls. One or more of the components of the kit may be lyophilized, in which case the kit may further include reagents suitable for reconstituting the lyophilized components.

The various components of the kit are optionally provided in suitable containers (e.g., microtiter plates) as needed. The kit may further include a container for holding or storing a sample (e.g., a urine, whole blood, plasma, or serum sample container or cartridge). Optionally, the kit may further include reaction vessels, mixing vessels, and reagents or other components to aid in the preparation of the test sample. The kit may further include one or more devices (e.g., syringes, pipettes, tweezers, measuring spoons, etc.) to aid in obtaining the test sample.

When the detectable label is at least one acridinium compound, the kit can include at least one acridinium-9-carboxamide, at least one acridinium-9-carboxylic acid aryl ester, or any combination thereof. When the detectable label is at least one acridinium compound, the kit can further include a source of hydrogen peroxide, such as a buffer, solution, and/or at least one basic solution. Optionally, the kit can include a solid phase, such as magnetic particles, beads, test tubes, microtiter plates, cuvettes, membranes, scaffold molecules, films, filter paper, disks, or chips.

Optionally, the kit can further include one or more components, alone or with instructions, for assaying the test sample for another analyte, which can be a biomarker, such as a biomarker for traumatic brain injury or disorder.

a. Application of Kits and Methods Kits (or components thereof) and methods for assessing or measuring the concentration of UCH-L1 and/or GFAP in a test sample by immunoassays as described herein are described, for example, in U.S. Pat. No. 5,063,081, U.S. Patent Application Publication Nos. 2003/0170881, 2004/0018577, 2005/0054078, and 2006/0160164, and are available, for example, from Abbott Laboratories, Inc. (Abbott Park, Ill.), under the name Abbott Point of Care (i-STAT® or i-STAT Alinity, Abbott Laboratories, Inc.). The devices can be adapted for use with a variety of automated and semi-automated systems (including systems in which the solid phase is particulate) commercially available from Abbott Laboratories, as well as systems described in U.S. Pat. Nos. 5,089,424 and 5,006,309 and commercially available, for example, from Abbott Laboratories (Abbott Park, Ill.) as the ARCHITECT® or Abbott Alinity series of devices.

Some differences between automated or semi-automated systems and non-automated systems (e.g., ELISA) include the substrate to which the first specific binding partner (e.g., analyte or capture antibody) is bound (which may affect sandwich formation and analyte reactivity) and the length and timing of the capture, detection, and/or optional wash steps. Non-automated formats such as ELISA may require relatively long incubation times with sample and capture reagent (e.g., about 2 hours), whereas automated or semi-automated formats (e.g., Abbott Laboratories ARCHITECT® and any successor platforms) may require relatively short incubation times (e.g., about 18 minutes for ARCHITECT®). Similarly, non-automated formats such as ELISA require relatively long incubation times for detection antibodies such as conjugate reagents (e.g., about 2 hours), whereas automated or semi-automated formats (e.g., ARCHITECT(R) and any successor platforms) can require relatively short incubation times (e.g., about 4 minutes for ARCHITECT(R) and any successor platforms).

Other platforms available from Abbott Laboratories include, but are not limited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, the entire disclosure of which is incorporated herein by reference), PRISM®, EIA (beads), and Quantum™ II, as well as other platforms. The assays, kits, and kit components can also be utilized in other formats, e.g., electrochemical or other handheld or point-of-care assay systems. As noted above, the present disclosure is applicable to, for example, commercially available point-of-care (Abbott Laboratories i-STAT®) electrochemical immunoassay systems that perform sandwich immunoassays. Immunosensors in disposable test devices and methods for their manufacture and operation are described, for example, in U.S. Pat. No. 5,063,081, U.S. Patent Application Publication Nos. 2003/0170881, 2004/0018577, 2005/0054078, and 2006/0160164, the entire teachings of which are incorporated herein by reference in their entirety in this regard.

For adapting the assay to the i-STAT® system, the following configuration is preferred: A microfabricated silicon chip is fabricated with a pair of gold amperometric working electrodes and a silver-silver chloride reference electrode. On one of the working electrodes, polystyrene beads (0.2 mm diameter) with immobilized capture antibodies are attached to a polymer coating of polyvinyl alcohol patterned over the electrode. The chip is embedded in an i-STAT® cartridge with a fluidics format suitable for immunoassays. A portion of the silicon chip is provided with one or more specific binding partners for UCH-L1 and/or GFAP, such as one or more UCH-L1 and/or GFAP antibodies (one or more monoclonal/polyclonal antibodies capable of binding to UCH-L1 and/or GFAP, or fragments thereof, mutants thereof, or fragments of mutants thereof) or one or more anti-UCH-L1 and/or GFAP DVD-Igs (or fragments thereof, mutants thereof, or fragments of mutants thereof capable of binding to UCH-L1 and/or GFAP), either of which may be detectably labeled. An aqueous reagent containing p-aminophenol phosphate is contained within the liquid pouch of the cartridge.

In operation, a sample from a subject suspected of having TBI is injected into the holding chamber of the test cartridge and the cartridge is inserted into the i-STAT® reader. A pump element in the cartridge pushes the sample into a conduit that houses the chip. The sample is contacted with the sensor, dissolving the enzyme conjugate into the sample. The sample is rocked between the sensors to facilitate sandwich formation for approximately 2-12 minutes. In the penultimate step of the assay, the sample is pushed into a waste chamber and excess enzyme conjugate and sample are washed off the sensor chip using a wash solution containing a substrate for the alkaline phosphatase enzyme. In the final step of the assay, the alkaline phosphatase label reacts with p-aminophenol phosphate to cleave the phosphate group, allowing the liberated p-aminophenol to be electrochemically oxidized at the working electrode. Based on the measured current, the reader can calculate the amount of UCH-L1 and/or GFAP in the sample using built-in algorithms and a factory-determined calibration curve.

The automated and semi-automated systems described herein for use in the methods of the present disclosure can utilize one or more computer programs, software, or algorithms to provide a decision (readout) of whether to perform a CT scan (e.g., based on a positive result) or not to perform a CT scan (e.g., based on a negative result). For example, the computer program or software (e.g., using an algorithm) can provide an interpretation (whether using one sample or two or more): (1) if the GFAP and UCH-L1 values are below the reference value (or cutoff value), the result is negative and a CT scan is not performed; or (2) if the GFAP and/or UCH-L1 are equal to or greater than the reference value (or cutoff value), the result is positive and a CT scan is performed. The computer program or software can also provide other appropriate interpretations, such as whether the reference value correlates to a positive head CT, the presence of an intracranial lesion, or control subjects without traumatic brain injury, whether a subject with TBI should be followed and/or treated for TBI, etc. Such computer programs or software are well known in the art.

The methods and kits described herein necessarily encompass other reagents and methods for performing immunoassays, including various buffers, such as those known in the art, and/or buffers that can be readily prepared or optimized for use as, for example, conjugate diluents and/or calibrator diluents for washing. One example of a conjugate diluent is ARCHITECT® Conjugate Diluent (Abbott Laboratories, Abbott Park, IL), which is utilized in certain kits and contains 2-(N-morpholino)ethanesulfonic acid (MES), salts, protein blockers, antimicrobial agents, and surfactants. One example of a calibrator diluent is ARCHITECT® Human Calibrator Diluent (Abbott Laboratories, Abbott Park, IL), which is utilized in certain kits and contains a buffer containing MES, other salts, protein blockers, and antimicrobial agents. Additionally, as described in U.S. Patent Application No. 61/142,048, filed December 31, 2008, the use of nucleic acid sequences linked to signal antibodies as signal amplifiers provides improved signal generation, for example in the i-STAT® cartridge format.

Although certain embodiments of the present application are advantageous when used to assess diseases such as traumatic brain injury, the assays and kits can optionally be used to assess UCH-L1 and/or GFAP in other diseases, disorders, and conditions as desired.

The assay methods can also be used to identify compounds that ameliorate disorders such as traumatic brain injury. For example, cells expressing UCH-L1 and/or GFAP can be contacted with a candidate compound. Using the assay methods described herein, the expression levels of UCH-L1 and/or GFAP in cells contacted with the compound can be compared to the expression levels in control cells.

The present invention has multiple aspects, which are illustrated by the following non-limiting examples.

Example 1 i-STAT® UCH-L1 Assay The i-STAT® UCH-L1 assay was used in a TBI patient population study of subjects with negative CT scans. A monoclonal antibody pair was used, including antibody A as the capture monoclonal antibody and antibodies B and C as the detection monoclonal antibodies. Antibody A is a typical anti-UCH-L1 antibody developed in-house at Abbott Laboratories (Abbott Park, IL). Antibodies B and C recognize various epitopes of UCH-L1 and enhance the detection of the antigen in the sample and were developed by Banyan Biomarkers (Alachua, Florida). Other antibodies developed in-house at Abbott Laboratories (Abbott Park, IL), or other commercially available antibodies, show or are expected to show comparable signal enhancement when used in various combinations as capture or detection antibodies. The UCH-L1 assay design was evaluated for key performance attributes. The cartridge configuration was antibody A (capture antibody)/antibody B+C (detection antibody), and the reagent conditions were 0.8% solids, 125 μg/mL Fab alkaline phosphatase cluster conjugate, and Sample Inlet Print: UCH-L1 standard. The assay time was 10-15 minutes (sample capture time 7-12 minutes).

Example 2 i-STAT® GFAP Assay The i-STAT® GFAP assay was used in a TBI patient population study. A monoclonal antibody pair was used, with antibody A as the capture monoclonal antibody and antibody B as the detection monoclonal antibody. Antibodies A and B are typical anti-GFAP antibodies developed in-house at Abbott Laboratories (Abbott Park, IL). Antibodies A and B bind to epitopes within the same GFAP digest. The GFAP assay design was evaluated for key performance attributes. The cartridge configuration was antibody A (capture antibody)/antibody B (detection antibody), and the reagent conditions were 0.8% solids, Fab alkaline phosphatase cluster conjugate 250 μg/mL, and Sample Inlet Print: GFAP specific. The assay time was 10-15 minutes (sample capture time 7-12 minutes).

Example 3: Assessment of GFAP and UCH-L1 levels in CT scan-negative subjects To evaluate blood levels of biomarkers such as GFAP and UCH-L1 in predicting computed tomography (CT)-negative TBI, blood samples were collected from adult (i.e., age 18 years or older) trauma subjects undergoing head CT scans. Biomarker concentrations were evaluated in subjects with negative CT scans (Marshall Classification = 1).

1A-D show ROC analysis of UCH-L1 or GFAP values correlated with Glasgow Coma Score (GCS) severity in subjects with negative CT scans for TBI. Samples were assessed within 12 hours of injury (FIGS. 1A, 1C) or within about 24 hours (i.e., within about 24.1 hours) of injury (FIGS. 1B, 1D). GFAP values are shown in FIG. 1A and FIG. 1B. UCH-L1 values are shown in FIG. 1C and FIG. 1D. Reference values for GFAP ranged from about 99 pg/mL to about 1674 pg/mL at 12 hours and from about 114 pg/mL to about 941 pg/mL at 24 hours. Reference values for UCH-L1 ranged from about 229 pg/mL to about 665 pg/mL at 12 hours and from about 162 pg/mL to about 314 pg/mL at 24 hours.

Figures 2A-F show ROC analyses of UCH-L1 or GFAP values correlated with loss of consciousness after injury in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 2A, 2D), 12 hours (Figures 2B, 2E), or about 24 hours (i.e., about 24.1 hours) (Figures 2C, 2F) of injury. GFAP values are shown in Figures 2A-2C. UCH-L1 values are shown in Figures 2D-F.

Figures 3A-F show ROC analysis of UCH-L1 or GFAP values correlated with MRI results in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 3A, 3D), 12 hours (Figures 3B, 3E), or within about 24 hours (i.e., within about 24.1 hours) (Figures 3C, 3F) of injury. GFAP values are shown in Figures 3A-3C. UCH-L1 values are shown in Figures 3D-F. Reference values for GFAP at about 4 hours were about 50 pg/mL to about 220 pg/mL, reference values for GFAP at 12 hours were about 76 pg/mL to about 326 pg/mL, and reference values for GFAP at 24 hours were about 76 pg/mL to about 383 pg/mL. The reference values for UCH-L1 at 4 hours were about 235 pg/mL to about 396 pg/mL, at 12 hours were about 210 pg/mL to about 285 pg/mL, and at 24 hours were about 150 pg/mL to about 169 pg/mL.

Figures 4A-F show ROC analyses of UCH-L1 or GFAP values correlated with post-traumatic amnesia (i.e., presence or absence of amnesia) in subjects with negative CT scans for TBI. Samples were assessed within 4 hours (Figures 4A, 4D), 12 hours (Figures 4B, 4E), or about 24 hours (i.e., about 24.1 hours) (Figures 4C, 4F) of injury. GFAP values are shown in Figures 4A-C. UCH-L1 values are shown in Figures 4D-F.

As will be apparent to those skilled in the art, other suitable modifications and applications of the disclosed methods described herein are readily applicable and reasonable, and may be implemented using appropriate equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Although the disclosure has been described in detail above, it will be more clearly understood by reference to the following examples. It should be noted that these examples are merely illustrative of some aspects and embodiments of the disclosure, and should not be construed as limiting the scope of the disclosure. The disclosures of all journal materials, U.S. patents, and publications referred to in this application are incorporated herein in their entirety.

The present invention has multiple aspects, which are illustrated by the non-limiting examples described herein.

It should be understood that the above detailed description and the accompanying drawings are merely illustrative and should not be taken as limiting the scope of the present invention, which is defined solely by the following claims and equivalents thereof.

Various changes and modifications of the embodiments disclosed herein will occur to those skilled in the art. Such changes and modifications may include, but are not limited to, those related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, and may be made without departing from the spirit and scope of the invention.

For completeness, various aspects of the invention are itemized below.
Section 1. An improvement to a method for aiding in the diagnosis and evaluation of a subject having or suspected to have a head injury, the method comprising the steps of: (1) performing, in a sample obtained from the subject within about 24 hours after an actual or suspected head injury, an assay for measuring or detecting a level of a biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample; and (2) performing a head computed tomography scan on the subject within a clinically relevant time frame, the improvement comprising diagnosing the subject as likely to have a traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and the head CT scan is negative for TBI.
Clause 2. The improvement of clause 1, further comprising the step of administering to said subject a treatment for TBI if the value of said biomarker is higher than a reference value and said imaging is negative for TBI.
Clause 3. The improvement of any one of clauses 1 or 2, wherein the reference value is correlated with a cut-off value related to: (a) a value in a subject suffering from a head injury; (b) the occurrence of TBI in a subject; (c) a stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) a positive MRI for TBI rather than negative; (f) the occurrence of amnesia in a subject (i.e., the presence or absence of amnesia); or (g) a severity of TBI in a subject.
Clause 4. The improvement of any one of clauses 1-3, wherein the sample is taken within about 0 to about 12 hours after an actual or suspected head injury, or within about 12 to about 24 hours after a suspected head injury.
Clause 5. The improvement according to any one of clauses 1 to 4, wherein the measurement of UCH-L1 levels is carried out by immunoassay or clinical chemistry test.
Clause 6. The improvement according to any one of clauses 1 to 5, wherein the measurement of GFAP levels is carried out by immunoassay or clinical chemistry test.
Clause 7. The improvement of any one of clauses 1 to 6, wherein the assay is performed using a point-of-care assay or a single molecule detection method.
Clause 8. The improvement of any one of clauses 1-7, wherein the sample is selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal specimen, and a nasopharyngeal specimen.
Clause 9. The improvement of any one of clauses 1 to 8, wherein the sample is obtained after the subject has suffered or may have suffered a head injury due to bodily shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma.
Clause 10. The improvement of any one of clauses 1-9, wherein the sample is obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a fire, a chemical, and a toxin.
Clause 11. The improvement of clause 10, wherein the chemical or toxin is mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, an adhesive, a gas, an organometallic, a drug of abuse, or one or more combinations thereof.
Clause 12. The improvement of any one of clauses 1 to 11, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.
Clause 13. The improvement of any one of clauses 1 to 12, wherein the method may be performed on any subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory values, the classification of the subject as having suffered a mild, moderate, severe, or moderate to severe traumatic brain injury, whether the subject has low, moderate or high UCH-L1, GFAP, or UCH-L1 and GFAP levels, and the timing of events which caused or may have caused the subject to suffer a head injury.
Clause 14. The improvement of any one of clauses 1 to 13, further comprising the step of following up on said subject.
Clause 15. A method for aiding in the diagnosis and evaluation of a subject who has experienced or may experience a head injury, said method comprising:
(1) an assay to measure or detect the level of a biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in a sample obtained from the subject within about 24 hours after actual or suspected head injury, and (2) a head computed tomography (CT) scan performed within a clinically relevant time frame, either simultaneously or sequentially;
b. If the value of the biomarker is higher than a reference value and the head CT scan is negative for TBI, diagnosing the subject as likely to have a traumatic brain injury (TBI).
Clause 16. The method of clause 15, further comprising the step of administering to said subject a treatment for TBI if the value of said biomarker is higher than a reference value and said CT scan is negative for TBI.
Clause 17. The method of any one of clauses 15 or 16, wherein the reference value is correlated with a cut-off value related to: (a) a value in a subject suffering from a head injury; (b) the occurrence of TBI in a subject; (c) a stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) a positive MRI for TBI rather than negative; (f) the occurrence of amnesia in a subject (i.e. the presence or absence of amnesia); or (g) a severity of TBI in a subject.
Clause 18. The method of any one of clauses 15-17, wherein the sample is taken within about 0 to about 12 hours after actual or suspected head injury, or within about 12 to about 24 hours after suspected head injury.
Clause 19. The method of any one of clauses 15 to 18, wherein the measurement of the UCH-L1 level is carried out by immunoassay or clinical chemistry test.
Clause 20. The method according to any one of clauses 15 to 19, wherein the measurement of the GFAP level is carried out by immunoassay or clinical chemistry test.
Clause 21. The method of any one of clauses 15 to 20, wherein the assay is performed using a point-of-care assay or a single molecule detection method.
Clause 22. The method of any one of clauses 15 to 21, wherein the sample is selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal specimen, and a nasopharyngeal specimen.
Clause 23. The method of any one of clauses 15 to 22, wherein the sample is obtained after the subject has suffered or may have suffered a head injury due to bodily shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma.
Clause 24. The method of any one of clauses 15 to 22, wherein the sample is obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a fire, a chemical, and a toxin.
Clause 25. The method of clause 24, wherein the chemical or toxin is mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, an adhesive, a gas, an organometallic, a drug of abuse, or one or more combinations thereof.
Clause 26. The method of any one of clauses 15 to 22, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.
Clause 27. The method of any one of clauses 15 to 22, wherein said method may be performed on any subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory values, the classification of the subject as having suffered a mild, moderate, severe, or moderate to severe traumatic brain injury, whether the subject has low, moderate or high UCH-L1, GFAP, or UCH-L1 and GFAP values, and the timing of events which caused or may have caused said subject to suffer a head injury.
Clause 28. The method of any one of clauses 15 to 27, further comprising the step of following up said subject.
Section 29. An improvement to a method of aiding in the diagnosis and evaluation of a subject having or possibly having a head injury, wherein the subject has a head computed tomography (CT) scan within a clinically relevant time frame of actual or suspected head injury that is negative for traumatic brain injury (TBI), the method comprising performing, concurrently or sequentially with the head CT, an assay to measure or detect in a sample obtained from the subject within about 24 hours after the actual or suspected head injury, a level of a biomarker in the sample comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, wherein the improvement comprises diagnosing the subject as likely to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value.
Section 30. An improvement to a method aiding in the diagnosis and evaluation of a subject having or possibly having a head injury, the method comprising: performing an assay to measure or detect in a sample obtained from the subject within about 24 hours after an actual or suspected head injury a level of a biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample, the improvement comprising diagnosing the subject as likely to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and a head computed tomography (CT) scan performed on the subject within a clinically relevant time frame is negative for TBI or no head CT scan has been performed on the subject.
Clause 31. The improvement of clause 30, further comprising the step of administering to the subject treatment for TBI if the value of the biomarker is higher than a reference value and, optionally, performing a head CT scan, the results of which are negative for TBI.
Clause 32. The improvement of any one of clauses 30 or 31, wherein the reference value is correlated with a cut-off value related to: (a) a value in a subject suffering from a head injury; (b) occurrence of TBI in a subject; (c) stage of TBI in a subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in a subject; (e) MRI positive for TBI rather than negative; (f) occurrence of amnesia in a subject (i.e. presence or absence of amnesia) or (g) severity of TBI in a subject.
Clause 33. The improvement of any one of clauses 30-32, wherein the sample is taken within about 0 to about 12 hours after actual or suspected head injury, or within about 12 to about 24 hours after actual or suspected head injury.
Clause 34. The improvement of any one of clauses 30 to 33, wherein measuring the level of UCH-L1 is carried out by immunoassay or clinical chemistry test.
Clause 35. The improvement of any one of clauses 30 to 34, wherein the measurement of GFAP levels is carried out by immunoassay or clinical chemistry test.
Clause 36. The improvement of any one of clauses 30 to 35, wherein the assay is performed using a point-of-care assay or a single molecule detection method.
Clause 37. The improvement of any one of clauses 30 to 36, wherein the sample is selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal specimen, and a nasopharyngeal specimen.
Clause 38. The improvement of any one of clauses 30 to 37, wherein the sample is obtained after the subject has experienced actual head injury due to bodily shaking, blunt impact from an external mechanical or other force resulting in closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma.
Clause 39. The improvement of any one of clauses 30-37, wherein the sample is obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a fire, a chemical, and a toxin.
Clause 40. The improvement of clause 39, wherein the chemical or toxin is mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, an adhesive, a gas, an organometallic, a drug of abuse, or one or more combinations thereof.
Clause 41. The improvement of any one of clauses 30 to 37, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof.
Clause 42. The improvement of any one of clauses 30 to 41, wherein the method may be performed on any subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory values, the classification of the subject as having suffered a mild, moderate, severe, or moderate to severe traumatic brain injury, whether the subject has low, moderate or high UCH-L1, GFAP, or UCH-L1 and GFAP levels, and the timing of events which caused or may have caused the subject to suffer a head injury.
Clause 43. The improvement of any one of clauses 30 to 42, further comprising the step of following up on said subject.
Clause 44. The improvement of any one of clauses 1 to 14, wherein the blood sample is a whole blood sample, a serum sample, or a plasma sample.
Clause 45. The method of any one of clauses 1 to 14 or 44, wherein the subject is a human subject.
Clause 46. The method of any one of clauses 15 to 28, wherein the blood sample is a whole blood sample, a serum sample, or a plasma sample.
Clause 47. The method of any one of clauses 15-28 or 46, wherein the subject is a human subject.
Clause 48. The improvement of any one of clauses 29 to 42, wherein the blood sample is a whole blood sample, a serum sample, or a plasma sample.
Clause 49. The improvement of any one of clauses 29 to 42 or 48, wherein the subject is a human subject.
Clause 50. The improvement of any one of clauses 29 to 49, wherein the reference value for GFAP is from about 30 pg/mL to about 1700 pg/mL and the reference value for UCH-L1 is from about 150 pg/mL to about 700 pg/mL.
Item 51. The improvement of item 50, wherein the reference value for GFAP is from about 90 pg/mL to about 1680 pg/mL and the reference value for UCH-L1 is from about 220 pg/mL to about 670 pg/mL.
Item 52. The improvement of item 51, wherein the reference value for GFAP is from about 110 pg/mL to about 950 pg/mL and the reference value for UCH-L1 is from about 160 pg/mL to about 320 pg/mL.

Claims (20)

An improvement to a method for aiding in the diagnosis and evaluation of a subject who has or may have suffered a head injury, the method comprising: (1) performing, in a sample obtained from the subject within about 24 hours after an actual or suspected head injury, an assay for measuring or detecting a level of a biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample; and (2) performing a head computed tomography (CT) scan on the subject within a clinically relevant time frame, the improvement comprising diagnosing the subject as likely to have a traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and the head CT scan is negative for TBI. An improvement to a method for aiding in the diagnosis and evaluation of a human subject who has suffered or may have suffered a head injury, the method comprising: performing an assay to measure or detect a level of a biomarker in a sample obtained from the subject within about 24 hours after an actual or suspected head injury, the biomarker comprising ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample; the improvement diagnosing the subject as likely to have traumatic brain injury (TBI) if the level of the biomarker is higher than a reference value and a head computed tomography (CT) scan performed on the subject within a clinically relevant time frame is negative for TBI or a head CT scan has not been performed on the subject. The improvement of claim 1 or 2, further comprising administering to the subject treatment for TBI if the value of the biomarker is higher than a reference value and, optionally, if a head CT scan is performed and is negative for TBI. The improvement of any one of claims 1 to 3, wherein the reference value is correlated with a cut-off value related to: (a) a value in a subject suffering from a head injury; (b) occurrence of TBI in the subject; (c) stage of TBI in the subject, such as mild, moderate, severe, or moderate-severe; (d) loss of consciousness in the subject; (e) MRI positive rather than negative for TBI; (f) occurrence of amnesia in the subject (i.e., the presence or absence of amnesia); or (g) severity of TBI in the subject. The improvement of any one of claims 1 to 4, wherein the sample is taken within about 0 to about 12 hours after an actual or suspected head injury, or within about 12 to about 24 hours after an actual or suspected head injury. The improvement according to any one of claims 1 to 5, wherein the measurement of UCH-L1 levels is performed by immunoassay or clinical chemistry test. The improvement according to any one of claims 1 to 6, wherein the measurement of the GFAP value is carried out by immunoassay or clinical chemistry test. The improvement of any one of claims 1 to 7, wherein the assay is performed using a point-of-care assay or a single molecule detection method. The improvement of any one of claims 1 to 8, wherein the sample is selected from the group consisting of a blood sample, a urine sample, a cerebrospinal fluid sample, a tissue sample, a body fluid sample, a saliva sample, an oropharyngeal sample, and a nasopharyngeal sample. The improvement of any one of claims 1 to 9, wherein the sample is obtained after the subject has experienced or may have experienced actual head injury due to bodily shaking, blunt impact from external mechanical or other forces resulting in closed or open head injury, one or more falls, an explosion or blast, or other type of blunt force trauma. The improvement of any one of claims 1 to 10, wherein the sample is obtained after the subject inhales or is exposed to a fire, a chemical, a toxin, or a combination of a fire, a chemical, and a toxin. The improvement of claim 11, wherein the chemical or toxin is mold, asbestos, a pesticide, an insecticide, an organic solvent, a paint, an adhesive, a gas, an organic metal, a drug of abuse, or one or more combinations thereof. The improvement of any one of claims 1 to 10, wherein the sample is obtained from a subject suffering from an autoimmune disease, a metabolic disorder, a brain tumor, hypoxia, a viral infection, a fungal infection, a bacterial infection, meningitis, hydrocephalus, or any combination thereof. The improvement according to any one of claims 1 to 13, wherein the method can be performed on any subject, regardless of factors selected from the group consisting of the subject's clinical condition, the subject's laboratory test values, the classification of the subject as having suffered mild, moderate, severe, or moderate to severe traumatic brain injury, whether the subject has low, medium, or high UCH-L1, GFAP, or UCH-L1 and GFAP values, and the timing of events that caused or may have caused the subject to suffer a head injury. The improvement according to any one of claims 1 to 14, further comprising a step of monitoring the subject. The improvement according to any one of claims 1 to 15, wherein the blood sample is whole blood, serum or plasma. The improvement according to any one of claims 1 to 16, wherein the subject is a human subject. The improvement according to any one of claims 1 to 17, wherein the reference value for GFAP is from about 30 pg/mL to about 1700 pg/mL and the reference value for UCH-L1 is from about 150 pg/mL to about 700 pg/mL. The improvement of claim 18, wherein the reference value for GFAP is from about 90 pg/mL to about 1680 pg/mL and the reference value for UCH-L1 is from about 220 pg/mL to about 670 pg/mL. The improvement of claim 19, wherein the reference value for GFAP is from about 110 pg/mL to about 950 pg/mL and the reference value for UCH-L1 is from about 160 pg/mL to about 320 pg/mL.

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